Note: Descriptions are shown in the official language in which they were submitted.
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TCR AND PEPTIDES
FIELD OF THE INVENTION
The present invention relates to T-cell receptors (TCRs) which bind to
peptides derived from
Wilms tumour 1 protein (WT1) when presented by a major histocompatibility
complex. In
this regard, the present invention relates to complementarity determining
regions (CDRs)
which specifically recognise WT1 peptides. The present invention further
relates to
immunogenic peptides derived from WT1.
BACKGROUND TO THE INVENTION
T cell receptor (TCR) gene therapy is based on the genetic transfer of high-
avidity tumour-
specific TCR genes into T lymphocytes, thus enabling the specific targeting of
the desired
tumour-associated antigens and leading to a less toxic and more specific and
effective
therapy. This approach has shown promise in clinical trials. One of the main
barriers limiting
the exploitation of TCR gene therapy for clinical treatment of cancers is the
lack of tumour-
specific T-cells and corresponding TCRs. Thus, the low availability of tumour-
specific TCRs
still remains an open issue limiting the broad exploitation of TCR-based
immunotherapeutic
approaches.
The majority of tumour-associated antigens (TAAs) are self antigens, thus T-
cells specific for
such molecules are either destroyed or anergized due to central and peripheral
tolerance.
Despite this, naturally occurring tumour-specific T-cells have been observed
in healthy
donors and patients, particularly in patients affected by hematological
malignancies, after
allogeneic hematopoietic stem cell transplantation (allo-HSCT) where
frequencies of tumor-
specific lymphocytes have been correlated with disease regression (Kapp, M. et
al. Bone
Marrow Transplantation 43,399-410 (2009); and Tyler, E.M. et al. Blood 121,308-
317
(2013)).
The choice of a tumor antigen to be targeted by immunotherapeutic approaches
is still a
matter of debate. Ideal TAAs are highly expressed on tumor cells while being
minimally
expressed in healthy tissue.
Wilms tumor 1 (WT1) is an intracellular protein encoding a zinc finger
transcription factor that
plays an important role in cell growth and differentiation (Yang, L. et al.
Leukemia 21, 868-
876 (2007)). WT1 is widely expressed on a variety of hematological and solid
tumors, while
showing limited expression on various healthy tissues (e.g. gonads, uterus,
kidney,
mesothelium, progenitor cells in different tissues). Recent evidence suggests
a role for WT1
in leukemogenesis and tumorigenesis.
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Several ongoing clinical trials rely on the generation of cytotoxic T
lymphocyte (CTL)
responses upon vaccination with WT1 peptides. However, despite the recognition
that WT1
is useful for immunotherapy, a small number of WT1 epitopes, which are
restricted to a
limited number of HLA alleles, are presently used for vaccination purposes (Di
Stasi, A. et al.
Front. lmmunol. (2015)). One such epitope is the WT1 126-134 epitope
(RMFPNAPYL; SEQ
ID NO: 71), which is presented by MHC encoded by the HLA-A*0201 allele (i.e.
the epitope
is HLA-A*0201 restricted).
HLA-A*0201 restricted epitopes and corresponding TCRs are of interest since
major
histocompatibility complex (MHC) having the HLA-A*0201 haplotype are expressed
in the
vast majority (60%) of the Caucasian population. Accordingly, TCRs that target
HLA-A*0201-
restricted WT1 epitopes are particularly advantageous since an immunotherapy
making use
of such TCRs may be widely applied.
The WT1 126-134 epitope has been widely studied in several trials, alone or in
combination
with additional tumor antigens. However, recent reports have highlighted a
major concern
regarding the processing of this particular epitope, which may impair its use
for
immunotherapy purposes. Notably, the WT1 126-134 epitope is more efficiently
processed
by the immunoproteasome compared with standard proteasomes (Jaigirdar, A. et
al. J
lmmunother. 39(3):105-16 (2016)), which leads to poor recognition of many HLA-
A*0201
tumour cell lines or primary leukemia cells that endogenously express WT1.
Thus, there remains a need for new WT1 epitopes, particularly those presented
by MHC with
prevalent HLA haplotypes (e.g. HLA-A*0201).
One naturally processed HLA-A*0201 restricted epitope that has been identified
is WT1 37-
45, which has the amino acid sequence VLDFAPPGA (SEQ ID NO: 72, see e.g.
Smithgall et
al 2001; Blood 98(11 Part 1): 121a). However, few TCR amino acid sequences,
particularly
CDR sequences, specific for this peptide sequence have been reported (Schmitt,
T.M. et al.
(2017) Nat Biotechnol 35: 1188-1195).
Accordingly, there remains a need for new WT1 epitopes, particularly those
restricted to
common HLA alleles and a need for new TCRs capable of binding to WT1 epitopes.
SUMMARY OF THE INVENTION
We have identified novel TCRs that bind to WT1 peptides when presented by an
MHC.
Further, we have determined the amino acid sequences of the TCRs, including
the amino
acid sequences of their CDR regions, which are responsible for binding
specificity for WT1.
Moreover, we have demonstrated that T-cells expressing TCRs according to the
invention
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specifically target and kill cells that overexpress the WT1 protein. In
addition, it has been
shown that TCRs of the present invention are restricted to MHC encoded by HLA
class I and
ll alleles common in the Caucasian population, such as HLA-A*0201, HLA-
B*38:01, HLA-
C*03:03 or HLA-C*07:02.
In one aspect, the invention provides a T-cell receptor (TCR), which binds to
a Wilms tumour
1 protein (WT1) peptide when presented by a major histocompatibility complex
(MHC),
wherein the TCR:
(i) comprises a CDR3a comprising the amino acid sequence of CASGGGADGLTF
(SEQ ID NO: 25) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR38 comprising the amino acid sequence of
CASGRGDTEAFF (SEQ ID NO: 30) or a variant thereof having up to three amino
acid substitutions, additions or deletions;
(ii) comprises a CDR3a comprising the amino acid sequence of CAMRTGGGADGLTF
(SEQ ID NO: 3) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR38 comprising the amino acid sequence of
CASSEAGLSYEQYF (SEQ ID NO: 8) or a variant thereof having up to three amino
acid substitutions, additions or deletions;
(iii) comprises a CDR3a comprising the amino acid sequence of
CILSTRVWAGSYQLTF
(SEQ ID NO: 14) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR38 comprising the amino acid sequence of
CATGQATQETQYF (SEQ ID NO: 19) or a variant thereof having up to three amino
acid substitutions, additions or deletions;
(iv) comprises a CDR3a comprising the amino acid sequence of
CAVIGGTDSWGKLQF
(SEQ ID NO: 36) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR38 comprising the amino acid sequence of
CASSQEEGAVYGYTF (SEQ ID NO: 41) or a variant thereof having up to three
amino acid substitutions, additions or deletions;
(v) comprises a CDR3a comprising the amino acid sequence of CAVIGGTDSWGKLQF
(SEQ ID NO: 36) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR38 comprising the amino acid sequence of
CATSREGLAADTQYF (SEQ ID NO: 52) or a variant thereof having up to three
amino acid substitutions, additions or deletions;
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(vi) comprises a CDR3a comprising the amino acid sequence of
CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof having up to three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino
acid sequence of CATSREGLAADTQYF (SEQ ID NO: 52) or a variant thereof having
up to three amino acid substitutions, additions or deletions;
(vii) comprises a CDR3a comprising the amino acid sequence of
CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof having up to three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino
acid sequence of CASSQEEGAVYGYTF (SEQ ID NO: 41) or a variant thereof
having up to three amino acid substitutions, additions or deletions;
(viii) comprises a CDR3a comprising the amino acid sequence of CAAPNDYKLSF
(SEQ
ID NO: 93) or a variant thereof having up to three amino acid substitutions,
additions
or deletions, and a CDR3[3 comprising the amino acid sequence of
CASSSGLAFYEQYF (SEQ ID NO: 98) or a variant thereof having up to three amino
acid substitutions, additions or deletions;
(ix) comprises a CDR3a comprising the amino acid sequence of CAAPNDYKLSF
(SEQ
ID NO: 93) or a variant thereof having up to three amino acid substitutions,
additions
or deletions, and a CDR3[3 comprising the amino acid sequence of
CASSQLSGRDSYEQYF (SEQ ID NO: 104) or a variant thereof having up to three
amino acid substitutions, additions or deletions;
(x) comprises a CDR3a comprising the amino acid sequence of CAVRDGGATNKLIF
(SEQ ID NO: 110) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR3[3 comprising the amino acid sequence of
CASSTLGGELFF (SEQ ID NO: 120) or a variant thereof having up to three amino
acid substitutions, additions or deletions;
(xi) comprises a CDR3a comprising the amino acid sequence of CLVGGYTGGFKTIF
(SEQ ID NO: 115) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR3[3 comprising the amino acid sequence of
CASSTLGGELFF (SEQ ID NO: 120) or a variant thereof having up to three amino
acid substitutions, additions or deletions;
(xii) comprises a CDR3a comprising the amino acid sequence of
CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof having up to
three amino acid substitutions, additions or deletions, and a CDR3[3
comprising the
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amino acid sequence of CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136) or a
variant thereof having up to three amino acid substitutions, additions or
deletions;
(xiii) comprises a CDR3a comprising the amino acid sequence of
CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof having up to
three amino acid substitutions, additions or deletions, and a CDR38 comprising
the
amino acid sequence of CATSWGLNEQYF (SEQ ID NO: 142) or a variant thereof
having up to three amino acid substitutions, additions or deletions;
(xiv) comprises a CDR3a comprising the amino acid sequence of CAATSRDDMRF (SEQ
ID NO: 131) or a variant thereof having up to three amino acid substitutions,
additions or deletions, and a CDR38 comprising the amino acid sequence of
CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136) or a variant thereof having up to
three amino acid substitutions, additions or deletions;
(xv) comprises a CDR3a comprising the amino acid sequence of CAATSRDDMRF (SEQ
ID NO: 131) or a variant thereof having up to three amino acid substitutions,
additions or deletions, and a CDR38 comprising the amino acid sequence of
CATSWGLNEQYF (SEQ ID NO: 142) or a variant thereof having up to three amino
acid substitutions, additions or deletions;
(xvi) comprises a CDR3a comprising the amino acid sequence of CALPDKVIF (SEQ
ID
NO: 148) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, and a CDR38 comprising the amino acid sequence of
CASSVSAGSTGELFF (SEQ ID NO: 158) or a variant thereof having up to three
amino acid substitutions, additions or deletions;
(xvii) comprises a CDR3a comprising the amino acid sequence of CAGLYATNKLIF
(SEQ
ID NO: 153) or a variant thereof having up to three amino acid substitutions,
additions or deletions, and a CDR38 comprising the amino acid sequence of
CASSVSAGSTGELFF (SEQ ID NO: 158) or a variant thereof having up to three
amino acid substitutions, additions or deletions;
(xviii) comprises a CDR3a comprising the amino acid sequence of CAAPNDYKLSF
(SEQ
ID NO: 93) or a variant thereof having up to three amino acid substitutions,
additions
or deletions, and a CDR38 comprising the amino acid sequence of CASSTLGGELFF
(SEQ ID NO: 120) or a variant thereof having up to three amino acid
substitutions,
additions or deletions;
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(xix) comprises a CDR3a comprising the amino acid sequence of CAVRDGGATNKLIF
(SEQ ID NO: 110) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR3[3 comprising the amino acid sequence of
CASSSGLAFYEQYF (SEQ ID NO: 98) or a variant thereof having up to three amino
acid substitutions, additions or deletions;
(xx) comprises a CDR3a comprising the amino acid sequence of CAVRDGGATNKLIF
(SEQ ID NO: 110) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR3[3 comprising the amino acid sequence of
CASSQLSGRDSYEQYF (SEQ ID NO: 104) or a variant thereof having up to three
amino acid substitutions, additions or deletions;
(xxi) comprises a CDR3a comprising the amino acid sequence of CLVGGYTGGFKTIF
(SEQ ID NO: 115) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR3[3 comprising the amino acid sequence of
CASSSGLAFYEQYF (SEQ ID NO: 98) or a variant thereof having up to three amino
acid substitutions, additions or deletions; or
(xxii) comprises a CDR3a comprising the amino acid sequence of CLVGGYTGGFKTIF
(SEQ ID NO: 115) or a variant thereof having up to three amino acid
substitutions,
additions or deletions, and a CDR3[3 comprising the amino acid sequence of
CASSQLSGRDSYEQYF (SEQ ID NO: 104) or a variant thereof having up to three
amino acid substitutions, additions or deletions.
In one embodiment, the TCR comprises the following CDR sequences:
(i) CDR1a - NSAFQY (SEQ ID NO: 23),
CDR2a - TYSSGN (SEQ ID NO: 24),
CDR3a - CASGGGADGLTF (SEQ ID NO: 25),
CDR1[3 - SGDLS (SEQ ID NO: 28),
CDR2[3 - YYNGEE (SEQ ID NO: 29), and
CDR3[3 - CASGRGDTEAFF (SEQ ID NO: 30),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(ii) CDR1a - TSDQSYG (SEQ ID NO: 1),
CDR2a - QGSYDEQN (SEQ ID NO: 2),
CDR3a - CAM RTGGGADGLTF (SEQ ID NO: 3),
CDR1[3 - SNHLY (SEQ ID NO: 6),
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CDR2[3 - FYNNEI (SEQ ID NO: 7), and
CDR3[3 - CASSEAGLSYEQYF (SEQ ID NO: 8),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(iii) CDR1a - TISGTDY (SEQ ID NO: 12),
CDR2a - GLTSN (SEQ ID NO: 13),
CDR3a - CILSTRVWAGSYQLTF (SEQ ID NO: 14),
CDR1[3 - KGHDR (SEQ ID NO: 17),
CDR2[3 - SFDVKD (SEQ ID NO: 18), and
CDR3[3 - CATGQATQETQYF (SEQ ID NO: 19),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(iv) CDR1a - DRGSQS (SEQ ID NO: 34),
CDR2a - IYSNGD (SEQ ID NO: 35),
CDR3a - CAVIGGTDSWGKLQF (SEQ ID NO: 36),
CDR1[3 - LGHNA (SEQ ID NO: 39),
CDR2[3 - YSLEER (SEQ ID NO: 40), and
CDR3[3 - CASSQEEGAVYGYTF (SEQ ID NO: 41),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(v) CDR1a - DRGSQS (SEQ ID NO: 34),
CDR2a - IYSNGD (SEQ ID NO: 35),
CDR3a - CAVIGGTDSWGKLQF (SEQ ID NO: 36),
CDR1[3 - LNHNV (SEQ ID NO: 50),
CDR2[3 - YYDKDF (SEQ ID NO: 51), and
CDR3[3 - CATSREGLAADTQYF (SEQ ID NO: 52),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(vi) CDR1a - NSASQS (SEQ ID NO: 45),
CDR2a - VYSSGN (SEQ ID NO: 46),
CDR3a - CVVPRGLSTDSWGKLQF (SEQ ID NO: 47),
CDR1[3 - LNHNV (SEQ ID NO: 50),
CDR2[3 - YYDKDF (SEQ ID NO: 51), and
CDR3[3 - CATSREGLAADTQYF (SEQ ID NO: 52),
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or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(vii) CDR1a ¨ NSASQS (SEQ ID NO: 45),
CDR2a - VYSSGN (SEQ ID NO: 46),
CDR3a ¨ CVVPRGLSTDSWGKLQF (SEQ ID NO: 47),
CDR1[3 - LGHNA (SEQ ID NO: 39),
CDR2[3 - YSLEER (SEQ ID NO: 40), and
CDR3[3 - CASSQEEGAVYGYTF (SEQ ID NO: 41),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(viii) CDR1a ¨ VSNAYN (SEQ ID NO: 91),
CDR2a - GSKP (SEQ ID NO: 92),
CDR3a ¨ CAAPNDYKLSF (SEQ ID NO: 93),
CDR1[3 - SEHNR (SEQ ID NO: 96),
CDR2[3 - FQNEAQ (SEQ ID NO: 97), and
CDR3[3 - CASSSGLAFYEQYF (SEQ ID NO: 98),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(ix) CDR1a ¨ VSNAYN (SEQ ID NO: 91),
CDR2a - GSKP (SEQ ID NO: 92),
CDR3a ¨ CAAPNDYKLSF (SEQ ID NO: 93),
CDR1[3 - SGHDN (SEQ ID NO: 102),
CDR2[3 - FVKESK (SEQ ID NO: 103), and
CDR3[3 - CASSQLSGRDSYEQYF (SEQ ID NO: 104),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(x) CDR1a ¨ VSGNPY (SEQ ID NO: 108),
CDR2a - YITGDNLV (SEQ ID NO: 109),
CDR3a ¨ CAVRDGGATNKLIF (SEQ ID NO: 110),
CDR1[3 - MNHEY (SEQ ID NO: 118),
CDR2[3 - SMNVEV (SEQ ID NO: 119), and
CDR3[3 - CASSTLGGELFF (SEQ ID NO: 120),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
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(xi) CDR1a ¨ NIATNDY (SEQ ID NO: 113),
CDR2a - GYKTK (SEQ ID NO: 114),
CDR3a ¨ CLVGGYTGGFKTIF (SEQ ID NO: 115),
CDR16 - MNHEY (SEQ ID NO: 118),
CDR26 - SMNVEV (SEQ ID NO: 119), and
CDR36 - CASSTLGGELFF (SEQ ID NO: 120),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xii) CDR1a ¨ SSVSVY (SEQ ID NO: 124),
CDR2a - YLSGSTLV (SEQ ID NO: 125),
CDR3a ¨ CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126),
CDR16 - SEHNR (SEQ ID NO: 134),
CDR26 - FQNEAQ (SEQ ID NO: 135), and
CDR36 - CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xiii) CDR1a ¨ SSVSVY (SEQ ID NO: 124),
CDR2a - YLSGSTLV (SEQ ID NO: 125),
CDR3a ¨ CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126),
CDR16 - LNHNV (SEQ ID NO: 140),
CDR26 - YYDKDF (SEQ ID NO: 141), and
CDR36 - CATS WGLNEQYF (SEQ ID NO: 142),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xiv) CDR1a ¨ DSASNY (SEQ ID NO: 129),
CDR2a - IRSNVGE (SEQ ID NO: 130),
CDR3a ¨ CAATSRDDMRF (SEQ ID NO: 131),
CDR16 - SEHNR (SEQ ID NO: 134),
CDR26 - FQNEAQ (SEQ ID NO: 135), and
CDR36 - CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xv) CDR1a ¨ DSASNY (SEQ ID NO: 129),
CDR2a - IRSNVGE (SEQ ID NO: 130),
CDR3a ¨ CAATSRDDMRF (SEQ ID NO: 131),
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CDR1[3 - LNHNV (SEQ ID NO: 140),
CDR2[3 - YYDKDF (SEQ ID NO: 141), and
CDR3[3 - CATS WGLNEQYF (SEQ ID NO: 142),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xvi) CDR1a ¨ TRDTTYY (SEQ ID NO: 146),
CDR2a - RNSFDEQN (SEQ ID NO: 147),
CDR3a ¨ CALPDKVIF (SEQ ID NO: 148),
CDR1[3 - SGDLS (SEQ ID NO: 156),
CDR2[3 - YYNGEE (SEQ ID NO: 157), and
CDR3[3 - CASSVSAGSTGELFF (SEQ ID NO: 158),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xvii) CDR1a ¨ SIFNT (SEQ ID NO: 151),
CDR2a - LYKAGEL (SEQ ID NO: 152),
CDR3a ¨ CAGLYATNKLIF (SEQ ID NO: 153),
CDR1[3 - SGDLS (SEQ ID NO: 156),
CDR2[3 - YYNGEE (SEQ ID NO: 157), and
CDR3[3 - CASSVSAGSTGELFF (SEQ ID NO: 158),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xviii) CDR1a ¨ VSNAYN (SEQ ID NO:91),
CDR2a - GSKP (SEQ ID NO: 92),
CDR3a ¨ CAAPNDYKLSF (SEQ ID NO: 93),
CDR1[3 - MNHEY (SEQ ID NO: 118),
CDR2[3 - SMNVEV (SEQ ID NO: 119), and
CDR3[3 - CASSTLGGELFF (SEQ ID NO: 120),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xix) CDR1a ¨ VSGNPY (SEQ ID NO: 108),
CDR2a - YITGDNLV (SEQ ID NO: 109),
CDR3a ¨ CAVRDGGATNKLIF (SEQ ID NO: 110),
CDR1[3 - SEHNR (SEQ ID NO: 96),
CDR2[3 - FQNEAQ (SEQ ID NO: 97), and
CDR3[3 - CASSSGLAFYEQYF (SEQ ID NO: 98),
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or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xx) CDR1a ¨ VSGNPY (SEQ ID NO: 108),
CDR2a - YITGDNLV (SEQ ID NO: 109),
CDR3a ¨ CAVRDGGATNKLIF (SEQ ID NO: 110),
CDR16 - SGHDN (SEQ ID NO: 102),
CDR26 - FVKESK (SEQ ID NO: 103), and
CDR36 - CASSQLSGRDSYEQYF (SEQ ID NO: 104),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xxi) CDR1a ¨ NIATNDY (SEQ ID NO: 113),
CDR2a - GYKTK (SEQ ID NO: 114),
CDR3a ¨ CLVGGYTGGFKTIF (SEQ ID NO: 115),
CDR16 - SEHNR (SEQ ID NO: 96),
CDR26 - FQNEAQ (SEQ ID NO: 97), and
CDR36 - CASSSGLAFYEQYF (SEQ ID NO: 98),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions;
(xxii) CDR1a ¨ NIATNDY (SEQ ID NO: 113),
CDR2a - GYKTK (SEQ ID NO: 114),
CDR3a ¨ CLVGGYTGGFKTIF (SEQ ID NO: 115),
CDR16 - SGHDN (SEQ ID NO: 102),
CDR26 - FVKESK (SEQ ID NO: 103), and
CDR36 - CASSQLSGRDSYEQYF (SEQ ID NO: 104),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions; or
(xxiii) CDR1a - DRGSQS (SEQ ID NO: 182),
CDR2a - IYSNGD (SEQ ID NO: 183),
CDR3a - CASGGGADGLTF (SEQ ID NO: 25),
CDR16 - SGDLS (SEQ ID NO: 28),
CDR26 - YYNGEE (SEQ ID NO: 29), and
CDR36 - CASGRGDTEAFF (SEQ ID NO: 30),
or variants thereof each having up to three amino acid substitutions,
additions or
deletions.
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In one embodiment, the TCR comprises:
(i) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 26
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 31 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(ii) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 4 or
a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 9 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(iii) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 15
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 20 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(iv) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 37
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 42 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(v) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 37
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 53 or a variant thereof having at least 75%,
at
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least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(vi) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 48
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 53 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(vii) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 48
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 42 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(viii) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 94
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 99 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(ix) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 94
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 105 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(x) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 111
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
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amino acid sequence of SEQ ID NO: 121 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xi) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 116
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 121 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xii) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 127
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 137 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xiii) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 127
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 143 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xiv) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 132
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 137 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xv) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 132
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
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least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 143 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xvi) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 149
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 159 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xvii) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 154
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 159 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xviii) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 94
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 121 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xix) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 111
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 99 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xx) an a chain variable domain comprising the amino acid sequence of SEQ
ID NO: 111
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
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least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 105 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xxi) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 116
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 99 or a variant thereof having at least 75%,
at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto;
(xxii) an a chain variable domain comprising the amino acid sequence of SEQ ID
NO: 116
or a variant thereof having at least 75%, at least 80%, at least 85%, at least
90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at
least 75%, sequence identity thereto; and a 13 chain variable domain
comprising the
amino acid sequence of SEQ ID NO: 105 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto; or
(xxiii) an a chain variable domain comprising the amino acid sequence selected
from the
group consisting of SEQ ID NO: 185, 190 or a variant thereof having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, preferably at least 75%, sequence identity
thereto; and a
13 chain variable domain comprising the amino acid sequence of SEQ ID NO: 31
or a
variant thereof having at least 75%, at least 80%, at least 85%, at least 90%,
at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at
least
75%, sequence identity thereto.
In one embodiment, the TCR comprises:
(i) an a chain comprising the amino acid sequence of SEQ ID NO: 27 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:
203 and variants of SEQ ID NOs: 32, 33 and 203 having at least 75%, at least
80%,
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at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(ii) an a chain comprising the amino acid sequence of SEQ ID NO: 5 or a
variant thereof
having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least
96%, at least 97%, at least 98%, or at least 99%, preferably at least 75%,
sequence
identity thereto; and a 13 chain comprising an amino acid sequence selected
from the
group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 195 and variants
of
SEQ ID NOs: 10, 11 and 195 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(iii) an a chain comprising the amino acid sequence of SEQ ID NO: 16 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
197 and variants of SEQ ID NOs: 21, 22 and 197 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(iv) an a chain comprising the amino acid sequence of SEQ ID NO: 38 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:
215 and variants of SEQ ID NOs: 43, 44 and 215 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(v) an a chain comprising the amino acid sequence of SEQ ID NO: 38 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:
217 and variants of SEQ ID NOs: 54, 55 and 217 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
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(vi) an a chain comprising the amino acid sequence of SEQ ID NO: 49 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:
217 and variants of SEQ ID NOs: 54, 55 and 217 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(vii) an a chain comprising the amino acid sequence of SEQ ID NO: 49 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:
215 and variants of SEQ ID NOs: 43, 44 and 215 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(viii) an a chain comprising the amino acid sequence of SEQ ID NO: 95 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 101 and
variants
of SEQ ID NOs: 100 and 101 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(ix) an a chain comprising the amino acid sequence of SEQ ID NO: 95 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 107 and
variants
of SEQ ID NOs: 106 and 107 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(x) an a chain comprising the amino acid sequence of SEQ ID NO: 112 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
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sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 122, SEQ ID NO: 123 and
variants
of SEQ ID NOs: 122 and 123 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xi) an a chain comprising the amino acid sequence of SEQ ID NO: 117 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 122, SEQ ID NO: 123 and
variants
of SEQ ID NOs: 122 and 123 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xii) an a chain comprising the amino acid sequence of SEQ ID NO: 128 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 139 and
variants
of SEQ ID NOs: 138 and 139 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xiii) an a chain comprising the amino acid sequence of SEQ ID NO: 128 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 145 and
variants
of SEQ ID NOs: 144 and 145 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
.. (xiv) an a chain comprising the amino acid sequence of SEQ ID NO: 133 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 139 and
variants
of SEQ ID NOs: 138 and 139 having at least 75%, at least 80%, at least 85%, at
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least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xv) an a chain comprising the amino acid sequence of SEQ ID NO: 133 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 145 and
variants
of SEQ ID NOs: 144 and 145 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xvi) an a chain comprising the amino acid sequence of SEQ ID NO: 150 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 160, SEQ ID NO: 161 and
variants
of SEQ ID NOs: 160 and 161 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xvii) an a chain comprising the amino acid sequence of SEQ ID NO: 155 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 160, SEQ ID NO: 161 and
variants
of SEQ ID NOs: 160 and 161 having at least 75%, at least 80%, at least 85%, at
least
90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at least 75%, sequence identity thereto;
(xviii) an a chain comprising the amino acid sequence of SEQ ID NO: 95 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 122, SEQ ID NO: 123 and
variants
of SEQ ID NOs: 122 and 123 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
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(xix) an a chain comprising the amino acid sequence of SEQ ID NO: 112 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 101 and
variants
of SEQ ID NOs: 100 and 101 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xx) an a chain comprising the amino acid sequence of SEQ ID NO: 112 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 107 and
variants
of SEQ ID NOs: 106 and 107 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xxi) an a chain comprising the amino acid sequence of SEQ ID NO: 117 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 101 and
variants
of SEQ ID NOs: 100 and 101 having at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
preferably at least 75%, sequence identity thereto;
(xxii) an a chain comprising the amino acid sequence of SEQ ID NO: 117 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 107 and
variants
of SEQ ID NOs: 106 and 107 having at least 75%, at least 80%, at least 85%, at
least
90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
preferably at least 75%, sequence identity thereto; or
(xxiii) (a) an a chain comprising an amino acid sequence selected from the
group
consisting of SEQ ID NOs: 186, 191, 198, 199, 200, 201, 202 and variants of
SEQ ID
NOs: 186, 191, 198, 199, 200, 201 and 202 having at least 75%, at least 80%,
at
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least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at
least 99%, preferably at least 75%, sequence identity thereto; and a 13 chain
comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof
having at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at
least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence
identity
thereto;
(b) an a chain comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 186, 191, 198, 199, 200, 201, 202 and variants of
SEQ ID
NOs: 186, 191, 198, 199, 200, 201 and 202 having at least 75%, at least 80%,
at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at
least 99%, preferably at least 75%, sequence identity thereto; and a 13 chain
comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof
having at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at
least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence
identity
thereto; or
(c) an a chain comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 186, 191, 198, 199, 200, 201, 202 and variants of
SEQ ID
NOs: 186, 191, 198, 199, 200, 201 and 202 having at least 75%, at least 80%,
at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at
least 99%, preferably at least 75%, sequence identity thereto; and a 13 chain
comprising the amino acid sequence of SEQ ID NO: 203 or a variant thereof
having
at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at
least 97%, at least 98%, or at least 99%, preferably at least 75%, sequence
identity
thereto.
(xxiv) an a chain comprising the amino acid sequence of SEQ ID NO: 194 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
195 and variants of SEQ ID NOs: 10, 11 and 195 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(xxv) an a chain comprising the amino acid sequence of SEQ ID NO: 196 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
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sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
197 and variants of SEQ ID NOs: 21, 22 and 197 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(xxvi) an a chain comprising the amino acid sequence of SEQ ID NO: 214 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:
215 and variants of SEQ ID NOs: 43, 44 and 215 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(xxvii) an a chain comprising the amino acid sequence of SEQ ID NO: 214 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:
217 and variants of SEQ ID NOs: 54, 55 and 217 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto;
(xxviii) an a chain comprising the amino acid sequence of SEQ ID NO: 216 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:
217 and variants of SEQ ID NOs: 54, 55 and 217 having at least 75%, at least
80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto; or
(xxix) an a chain comprising the amino acid sequence of SEQ ID NO: 216 or a
variant
thereof having at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at
least 96%, at least 97%, at least 98%, or at least 99%, preferably at least
75%,
sequence identity thereto; and a 13 chain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:
215 and variants of SEQ ID NOs: 43, 44 and 215 having at least 75%, at least
80%,
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at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or
at least 99%, preferably at least 75%, sequence identity thereto.
In one embodiment, the TCR of the invention binds to a Wilms tumour 1 protein
(WT1)
peptide when presented by a major histocompatibility complex (MHC), wherein
the WT1
peptide comprises an amino acid sequence selected from the group consisting of
GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and
CMTWNQMNLGATLKG (SEQ ID NO: 87) and variants thereof each having up to three
amino acid substitutions, additions or deletions.
In another aspect, the invention provides a T-cell receptor (TCR), which binds
to a Wilms
tumour 1 protein (WT1) peptide when presented by a major histocompatibility
complex
(MHC), wherein the WT1 peptide comprises an amino acid sequence selected from
the
group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID
NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87) and variants thereof each having
up
to three amino acid substitutions, additions or deletions.
In one embodiment, the TCR binds to an MHC I and/or MHC II peptide complex.
In one embodiment, the TCR is restricted to a human leukocyte antigen (H LA)
allele. In one
embodiment, the TCR is restricted to a HLA-A, HLA-B or a HLA-C allele. In one
embodiment, the TCR is restricted to HLA-A*02:01, HLA-B*38:01, HLA-C*03:03 or
HLA-
C*07:02.
In one embodiment, the TCR is restricted to HLA-A*02:01. In one embodiment,
the TCR is
restricted to HLA-B*38:01. In one embodiment, the TCR is restricted to HLA-
C*03:03. In one
embodiment, the TCR is restricted to HLA-C*07:02.
In one embodiment, a TCR of the present invention is restricted to a HLA-C
allele. In one
embodiment, a TCR of the present invention is restricted to a HLA-C allele
selected from the
group consisting of HLA-C*07:01, HLA-C*03:04, HLA-C*04:01, HLA-C*05:01, HLA-
C*06:02
and HLA-C*07:02.
In one embodiment, the TCR comprises one or more mutations at the a chain/[3
chain
interface, such that when the a chain and the 13 chain are expressed in a T-
cell, the
frequency of mispairing between said chains and endogenous TCR a and 13 chains
is
reduced.
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In one embodiment, the TCR comprises one or more mutations at the a chain/6
chain
interface, such that when the a chain and the 13 chain are expressed in a T-
cell, the level of
expression of the TCR a and 13 chains is increased.
In one embodiment, the one or more mutations introduce a cysteine residue into
the
constant region domain of each of the a chain and the 13 chain, wherein the
cysteine
residues are capable of forming a disulphide bond between the a chain and the
13 chain.
In one embodiment, the one or more mutations are at amino acid positions
selected from
those disclosed in Table 1 of Boulter, J.M et al. (2003) Protein Engineering
16: 707-711.
In one embodiment, the TCR comprises one or more mutations to remove one or
more N-
glycosylation sites (see, for example, Kuball, J et al. (2009) J Exp Med 206:
463-75).
Preferably, the N-glycosylation sites are in the TCR constant domains. In one
embodiment,
the mutation is a substitution of the amino acid N in an N-X-S/T motif with
the amino acid Q.
For example, the substitution may occur at one or more of the positions: TCR
alpha constant
gene position 36, 90 or 109; and/or TCR beta constant gene position 85.6.
Preferably, the
substitution is at position 36 of the TCR alpha constant gene.
In one embodiment, the TCR comprises a murinised constant region.
In one embodiment, the TCR is a soluble TCR.
In another aspect, the invention provides an isolated polynucleotide encoding
the a chain of
a T-cell receptor (TCR) according to the invention, and/or the 13 chain of a
TCR according to
the invention.
In one embodiment, the polynucleotide encodes the a chain linked to the 13
chain.
In one embodiment, the isolated polynucleotide further encodes one or more
short interfering
RNA (siRNA) or other agents capable of reducing or preventing expression of
one or more
endogenous TCR genes.
In another aspect, the invention provides a vector comprising a polynucleotide
according to
the invention. In one embodiment, the vector comprises a polynucleotide, which
encodes
one or more CD3 chains, CD8, a suicide gene and/or a selectable marker.
In another aspect, the invention provides a cell comprising a TCR of the
invention, a
polynucleotide of the invention or a vector of the invention.
In one embodiment, the cell further comprises a vector which encodes one or
more CD3
chains, CD8, a suicide gene and/or a selectable marker.
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In one embodiment, the cell is a T-cell, a lymphocyte, or a stem cell,
optionally wherein the
T-cell, the lymphocyte, or the stem cell is selected from the group consisting
of CD4 cells,
CD8 cells, naive T-cells, memory stem T-cells, central memory T-cells, double
negative T-
cells, effector memory T-cells, effector T-cells, Th0 cells, Tc0 cells, Th1
cells, Tc1 cells, Th2
cells, Tc2 cells, Th17 cells, Th22 cells, gamma/delta T-cells, natural killer
(NK) cells, natural
killer T (NKT) cells, cytokine-induced killer (CIK) cells, hematopoietic stem
cells and
pluripotent stem cells.
In one embodiment, the cell is a T-cell which has been isolated from a
subject.
In one embodiment, an endogenous gene encoding a TCR a chain and/or an
endogenous
gene encoding a TCR 13 chain in the cell is disrupted, preferably such that
the endogenous
gene encoding a TCR a chain and/or the endogenous gene encoding a TCR 13 chain
is not
expressed. In one embodiment, the endogenous gene encoding a TCR a chain
and/or the
endogenous gene encoding a TCR 13 chain is disrupted by insertion of an
expression
cassette comprising a polynucleotide sequence encoding the TCR of the
invention. In one
embodiment, one or more endogenous genes encoding an MHC is disrupted,
preferably
wherein the cell is a non-alloreactive universal T-cell. In one embodiment, an
endogenous
gene involved in persistence, expansion,
activity, resistance to
exhaustion/senescence/inhibitory signals, homing capacity, or other T-cell
functions is
disrupted, preferably wherein the endogenous gene involved in persistence,
expansion,
activity, resistance to exhaustion/senescence/inhibitory signals, homing
capacity, or other T-
cell functions is selected from the group consisting of PD1, TIM3, LAG3, 284,
KLRG1,
TGFbR, CD160, TIGIT, CTLA4 and CD39. In one embodiment, the endogenous gene
involved in persistence, expansion, activity, resistance to
exhaustion/senescence/inhibitory
signals, homing capacity, or other T-cell functions is disrupted by
integration of an
expression cassette, wherein the expression cassette comprises a
polynucleotide sequence
encoding a TCR of the invention.
In another aspect, the invention provides a method of preparing a cell, which
comprises the
step of introducing the vector of the invention into a cell in vitro, ex vivo
or in vivo, for
example by transfection or transduction.
In another aspect, the invention provides a method of preparing a cell, which
comprises the
step of transducing a cell in vitro, ex vivo or in vivo with one or more
vectors of the invention.
In one embodiment, the cell to be transduced with the one or more vectors is
selected from
the group consisting of T-cells, lymphocytes or stem cells, such as
hematopoietic stem cells
or induced pluripotent stem cells (iPS), optionally the T-cell, the lymphocyte
or the stem cell
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may be selected from the group consisting of CD4 cells, CD8 cells, Th0 cells,
Tc0 cells, Th1
cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma/delta T-
cells, natural
killer (NK) cells, natural killer T (NKT) cells, double negative T-cells,
naive T-cells, memory
stem T-cells, central memory T-cells, effector memory T-cells, effector T
cells, cytokine-
induced killer (CIK) cells, hematopoeitic stem cells and pluripotent stem
cells.
In one embodiment, the method comprises the step of T-cell editing, which
comprises
disrupting an endogenous gene encoding a TCR a chain and/or an endogenous gene
encoding a TCR 13 chain with an artificial nuclease, preferably wherein the
artificial nuclease
is selected from the group consisting of zinc finger nucleases (ZFNs),
transcription activator-
like effector nucleases (TALENs) and CRISPR/Cas systems.
In one embodiment, the method comprises the step of T-cell editing, which
comprises
disrupting an endogenous gene encoding a TCR a chain and/or an endogenous gene
encoding a TCR 13 chain with an artificial nuclease, preferably wherein the
artificial nuclease
is selected from the group consisting of zinc finger nucleases (ZFNs),
transcription activator-
.. like effector nucleases (TALENs) and CRISPR/Cas systems.
In one embodiment, the method comprises the step of targeted integration of an
expression
cassette into the endogenous gene encoding the TCR a chain and/or the
endogenous gene
encoding the TCR 13 chain disrupted by the artificial nuclease, wherein the
expression
cassette comprises a polynucleotide sequence encoding the TCR of the invention
or a
.. polynucleotide sequence of the invention.
In one embodiment, the method comprises the step of disrupting one or more
endogenous
genes encoding an MHC, preferably wherein the cell prepared by the method is a
non-
alloreactive universal T-cell.
In one embodiment, the method comprises the step of disrupting one or more
endogenous
MHC genes, preferably wherein the cell prepared by the method is a non-
alloreactive
universal T-cell.
In one embodiment, the method comprises the step of disrupting one or more
endogenous
genes to modify the persistence, expansion, activity, resistance to
exhaustion/senescence/inhibitory signals, homing capacity, or other T-cell
functions,
preferably wherein the method comprises the step of targeted integration of an
expression
cassette into an endogenous gene involved in persistence, expansion, activity,
resistance to
exhaustion/senescence/inhibitory signals, homing capacity, or other T-cell
functions
disrupted by an artificial nuclease, wherein the expression cassette comprises
a
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polynucleotide sequence encoding the TCR of the invention, preferably wherein
the
endogenous gene is selected from the group consisting of PD1, TIM3, LAG3, 284,
KLRG1,
TGFbR, CD160, TIGIT, CTLA4 and CD39.
In another aspect, the invention provides the cell of the invention or a cell
prepared by the
method of the invention for use in adoptive cell transfer, preferably adoptive
T-cell transfer,
optionally wherein the adoptive T-cell transfer is allogenic adoptive T-cell
transfer,
autologous adoptive T-cell transfer, or universal non-alloreactive adoptive T-
cell transfer.
In another aspect, the invention provides a chimeric molecule comprising the
TCR of the
invention, or a portion thereof, conjugated to a non-cellular substrate, a
toxin and/or an
antibody. In one embodiment, the non-cellular substrate is selected from the
group
consisting of nanoparticles, exosomes and other non-cellular substrates.
In another aspect, the invention provides the TCR of the invention, the
isolated
polynucleotide of the invention, the vector of the invention, the cell of the
invention, a cell
prepared by the method of the invention, or the chimeric molecule of the
invention for use in
therapy.
In another aspect, the invention provides the TCR of the invention, the
isolated
polynucleotide of the invention, the vector of the invention, the cell of the
invention, a cell
prepared by the method of the invention, or the chimeric molecule of the
invention for use in
treating and/or preventing a disease associated with expression of WT1.
In another aspect, the invention provides a T-cell genetically engineered
(e.g. genetically
edited) to modify the persistence, expansion, activity, resistance to
exaustion/senescence/inhibitory signals, homing capacity or other T cell
functions, wherein
the T-cell expresses a TCR a chain of the invention and/or a TCR 13 chain of
the invention.
In another aspect, the invention provides a T cell genetically engineered
(e.g. genetically
edited) by a protocol which comprises the step of targeted integration of an
expression
cassette into an endogenous gene involved in persistence, expansion, activity,
resistance to
exhaustion/senescence/inhibitory signals, homing capacity or other T-cell
functions disrupted
by an artificial nuclease, wherein the expression cassette comprises a
polynucleotide
sequence encoding TCR a chain of the invention and/or a TCR 13 chain of the
invention.
In another aspect, the invention provides a method for treating and/or
preventing a disease
associated with expression of WT1, which comprises the step of administering
the TCR of
the invention, the isolated polynucleotide of the invention, the vector of the
invention, the cell
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of the invention, a cell prepared by the method of the invention, or the
chimeric molecule of
the invention to a subject in need thereof.
In one embodiment, the disease associated with expression of WT1 is a
proliferative
disorder. Preferably, the proliferative disorder is a hematological malignancy
or a solid
tumor. Preferably, the hematological malignancy is selected from the group
consisting of
acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic
leukemia,
myelodisplastic syndromes, lymphoma, multiple myeloma, non Hodgkin lymphoma,
and
Hodgkin lymphoma. Preferably, the solid tumor is selected from the group
consisting of lung
cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer,
cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers,
synovial
sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer,
neuroblastoma,
rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft
tissue
sarcoma, urothelial cancer, biliary cancer, glioblastoma, cervical cancer,
mesothelioma and
colorectal cancer.
In a preferred embodiment, the disease associated with expression of WT1 is
acute myeloid
leukemia (AML).
In another preferred embodiment, the disease associated with expression of WT1
is chronic
myeloid leukemia (CML).
In another aspect, the invention provides an isolated immunogenic WT1 peptide
comprising
an amino acid sequence selected from the group consisting of GAQYRIHTHGVFRGI
(SEQ
ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID
NO: 87) and variants thereof each having up to three amino acid substitutions,
additions or
deletions.
DESCRIPTION OF THE DRAWINGS
FIGURE 1
Evaluation over time of WT1-specific T cell expansion in 4 healthy donors.
Peripheral
blood mononuclear cells of four HDs were repetitively stimulated with the WT1
pool-137
(HD12, a) or with the WT1 HLA-A*02:01 pool (HD13-HD15, b-d). Enrichment of
antigen-
responding T cells was assessed by measuring IFNy secretion and CD107a
production in a
6 hour co-culture with autologous antigen-presenting cells (APCs) pulsed with
a pool derived
from WT1 protein. In each test, T cells unstimulated, T cells in co-culture
with APCs loaded
with an unrelated peptide pool and T cells stimulated with Phorbol-12-
myristate-13-acetate
(PMA) and lonomycin (not shown) were included as controls. Dot plots indicate
the results of
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the intracellular staining for IFNy production and CD107a exposure on cell
surface either at a
single time point (a, b, d) or over the culture timeframe (c). HD, healthy
donor; WT1, Wilms
Tumor 1; PMA, Phorbol 12-myristate 13-acetate; IFNy, interferon-y; S,
stimulation.
FIGURE 2
Identification of the WTI subpools and peptides recognised by expanded T
lymphocytes isolated from each HD. The assessment of the peptides inducing an
immune
response in T cells isolated from HDs was performed with a co-culture of T
cells with APCs
loaded with 24 subpools (SPs; HD12) or 11 peptides (HD13 and HD14).
Additionally,
negative (T-cells unstimulated and T-cells co-cultured with APCs loaded with
an unrelated
peptide pool or unrelated peptide) and positive (T-cells cultured in the
presence of PMA and
lonomycin) controls were included in the experimental setting (not shown).
Evaluation of
IFNy secretion and CD107a expression was performed by cytofluorimetric
analysis.
Representative dot plots relative to the co-culture of the T-cells with APCs
loaded with the
subpools and indicating the expression of IFNy and CD107a are reported.
Dominant
responses were observed for subpools 7 and 21 in HD12 (a, b), peptide 13 for
HD13 and
HD14 (c-e). HD, healthy donor; SP, subpools; WT1, Wilms Tumor 1; APC, antigen-
presenting cells; PMA, Phorbol 12-myristate 13-acetate; IFNy, interferon-y.
FIGURE 3
Epitope specificity of the WT1-reactive T cells generated by sensitisation
with the
pooled peptides. In order to validate the WT1 immunogenic peptides, T-cells
expanded
from HD12 were co-cultured for 6 hours in the presence of APCs loaded with the
peptide
identified after deconvolution of the mapping grid (a) and with at least one
unrelated peptide
as a negative control. Additionally, negative (T cells unstimulated) and
positive (T cells
cultured in the presence of PMA and lonomycin) controls were included in the
experimental
setting (not shown). Dot plots show for each HD the results of the
intracellular staining for
IFNy and surface CD107a. Enrichment of CD107a and IFNy positive cells was
observed for
T-cells co-cultured with peptide 103 and not for the unrelated peptide (b).
WT1, Wilms
Tumor 1; APC, antigen-presenting cells; PMA, 2; Phorbol 12-myristate 13-
acetate; IFNy,
interferon-y.
FIGURE 4
In silk prediction of HLA-peptide binding for different HDs. HLA typing
results for
HD12-HD14 (a). Results of the in silico prediction performed with the
NetMHC4.0 pan
algorithm are reported (b, HD12; c, HD13; d, HD14; e, HD15). In grey are
highlighted the
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peptides identified as strong binders by the algorithm. HD, healthy donor;
HLA, human
leukocyte antigen.
FIGURE 5
HLA-restriction assessment of the WTI immunogenic epitopes identified. To
determine
the HLA restriction element for HD12 we co-cultured T cells with different
antigen presenting
EBV-BLCL cell lines, each one harboring a specific HLA allele of interest
shared with HD12
and pulsed with peptide 103 or with an unrelated peptide as a control (a). For
HD13 and
HD14, WT1-specific T cells were co-cultured with T2 cells harbouring the HLA-
A*02:01
restriction element and pulsed with peptide 13 or with an unrelated peptide as
a control (b
and c, respectively). As readout we determined the expression of the CD107a
marker and
the secretion of IFNy. HD, healthy donor; WT1, Wilms Tumor 1; IFNy, interferon-
y; HLA,
human leukocyte antigen; EBV, Epstein-Barr virus; BLCL, B lymphoblastoid cell
line.
FIGURE 6
Immunogenic peptides are naturally processed. T cells isolated from HD13 (a)
and HD14
(b) were co-cultured with primary AML blasts from 3 different patients
(indicated as
pAML#15, pAML#16.1; pAML#16.2) expressing WT1 at high levels and harboring the
HLA-
A*02:01 restriction element. As a control, we included co-cultures of the
blasts with unrelated
T cells. We evaluated the percentage of Caspase 3 (Cas3) expression in target
(T) living
cells upon 6 hour co-culture with effector (E) T cells at different E:T
ratios. Cas3 values
.. obtained in the control conditions were subtracted from the Cas3 values
obtained from the
co-cultures of primary blasts with HD13 and HD14 T cells. pAML, primary blasts
of acute
myeloid leukemia patients; HD, healthy donor; HLA, human leukocyte antigen;
WT1, Wilms
Tumor 1.
FIGURE 7
WT1-specific TCR VI3 profile characterisation. WT1-specific T cells isolated
from HDs
were stained in order to quantitatively determine the TCR 13-chain variable
region (V13)
repertoire by FACS analysis. Results indicated the expression of a highly
dominant V13 gene
in HD12 and HD14, whereas for HD13 a clear enrichment of a defined V13 was not
detected.
For HD15 it was not possible to perform the V13 immunoprofiling analysis due
to a reduced
cell fitness. TRBV, T cell receptor variable beta chain; HD, healthy donor;
FACS,
fluorescence activated cell sorter.
FIGURE 8
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Clonal tracking of WT1-specific TCRs. TCRa8 sequencing was performed on HD RNA
at
different time points over the culture timeframe. Sequencing results indicated
the presence
of predominant clonotypes for HD12 (a), HD13 (b), HD14 (c) and HD15 (d). Bar
charts depict
the ten most predominant CDR3 amino acid sequences identified at each time
point (e.g. S4
corresponds to the sequencing results obtained following the 4th round of
stimulation). For
each bar, starting from the x-axis, the bottom segment represents the most
predominant
CDR3 sequence. The next nine most predominant sequences are stacked above the
bottom
segment and are ordered by decreasing frequency going upwards. The remaining
sequences are grouped together in the top segment. CDR3, complementarity
determining
region 3; S, stimulation; HD, healthy donor; S, stimulation; HLA, human
leukocyte antigen; P,
peptide; RNA, ribonucleic acid.
FIGURE 9
Functional avidity of TCR derived from HD12. T cells from 3 different healthy
donors were
transduced with a lentiviral vector encoding TCR isolated from HD12 upon knock-
out of the
endogenous a and 13 chains. a) Expression of the HD12-derived TCR was assessed
by V13
staining before and after V13-enrichment. b) Functional avidity of HD12-
derived TCR. We co-
cultured effector cells with EBV cell line pulsed either with the NYESO-1
peptide as control
or with decreasing concentrations of the peptide 103 (40 pg-0.4 pg, as
indicated on the x-
axis). Upon 6 hours of co-culture, results showed the ability of HD12-edited T
cells to
recognise target cells in presence of at least 0.4 pg of the cognate peptide.
No recognition of
the unrelated peptide was measured. As a readout we evaluated by flow
cytometry the
expression of the CD107a on the CD8 T lymphocytes. TCR, T cell receptor; HD,
healthy
donor; PBMC, peripheral blood mononuclear cell; NYESO-1, New York esophageal
squamous cell carcinoma 1.
FIGURE 10
Functional validation of HD13-derived TCR. T cells from one healthy donor were
transduced with a lentiviral vector encoding HD13-derived TCR. a) Recognition
of WT1 pool
by TCR isolated from HD13. We co-cultured effector cells with T2 cell line
pulsed either with
WT1 pool or an unrelated pool as control. Additionally, negative (T cells
unstimulated) and
positive (T cells cultured in the presence of PMA and lonomycin) controls were
included in
the experimental setting. Upon 6 hours of co-culture, T cells transduced with
HD13-derived
TCR to specifically recognise target cells pulsed with WT1 pool as assessed by
measuring
IFNy secretion on CD8 T cells. b) T cells were tested in co-culture with T2
cells pulsed with
WT1-derived SPs 1 and 14, both containing peptide 13, or SP 6 as a negative
control.
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Results showed the ability of effector cells to specifically recognise SP1 and
14 as evaluated
by measuring IFNy secretion and the expression of CD107a on CD8 T cells. HD,
healthy
donor; TCR, T cell receptor; WT1, Wilms tumor 1; SP, subpool; PMA, 2; Phorbol
12-
myristate 13-acetate.
FIGURE 11
Functional validation of TCR isolated from HD14. T cells isolated from one
healthy donor
were transduced with a lentiviral vector encoding HD14-derived TCRs (TRAV12-
2*01 WT
and TRAV12-2*02 WT). a) Transduction efficiency of HD14-transfer T cells
expressed as V13
expression on CD4 and CD8 T lymphocytes. b) Recognition of WT1 pool by HD14-
derived
TCRs. We co-cultured effector cells with T2 cell line pulsed either with WT1
pool or an
unrelated pool as a control. Upon 6 hours of co-culture, results showed the
ability of HD14-
transfer T cells to specifically recognise target cells pulsed with WT1 pool
as measured by
evaluating IFNy secretion on CD8 T cells. c) HD14-derived T cells recognise
specific SPs
containing peptide 13. T cells were tested in co-culture with T2 cells pulsed
with WT1-
derived SPs 1 and 14, both containing peptide 13, or SP 6 as a negative
control. Results
showed the ability of effector cells to specifically recognise SP1 and 14 as
evaluated by
assessing the expression of CD107a and IFNy secretion on CD8 T cells. HD,
healthy donor;
TCR, T cell receptor; WT1, Wilms tumor 1; SP, subpool.
FIGURE 12
TCRs derived from HD14 recognise primary AML blasts. TCR-edited T cells from
one
healthy donor were transduced with a lentiviral vector encoding HD14-derived
TCRs (TRAV
12-2*02 WT and TRAV 12-2*02 mut). a) Transduction efficiency of HD14 TCRs was
assessed by V13 expression on CD4 and CD8 T cells. b) Edited T cells
transduced with
HD14 TCR TRAV 12-2*02 WT, TRAV12-2*02 mut or an unrelated TCR were co-cultured
with patient-derived primary AML blasts expressing high levels of WT1 and the
HLA-A02*01
restriction element. To assess viability of blasts we included conditions of
target cells without
T lymphocytes. We evaluated the percentage of Caspase 3 (Cas3) expression in
target (T)
living cells upon 6 hour co-culture with effector (E) T cells at different E:T
ratios (as indicated
in the figure). Cas3 values obtained in the control conditions (either T cells
transduced with
unrelated TCR or blasts alone) were subtracted from the Cas3 values obtained
from the co-
cultures of primary blasts with T cells harbouring HD14-derived TCRs. pAML,
primary blasts
of acute myeloid leukemia patients; HD, healthy donor; HLA, human leukocyte
antigen;
WT1, Wilms Tumor 1.
FIGURE 13
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Identification of WT1-specific T cells by dextramer staining. Dot plots
indicate the
results of Dextramer staining at different time points upon T cell sorting
(using an APC-
labelled dextramer specific for the WT1 VLDFAPPGA peptide) and stimulation
(Patient 1, a)
or at a single time point (b, Patients 2 and 3). WT1, Wilms Tumor 1.
FIGURE 14
Graphs showing results of TCR sequencing of enriched WT1-specific T-cells. T-
cells
isolated from each patient and sorted based on the positivity for WT1
dextramer staining
were characterised by TCR a13 sequencing. Sequencing results indicated the
presence of
predominant clonotypes for Patient 1 (a, b), Patient 2 (c) and Patient 3 (d).
Bar charts depict
the ten most predominant CDR3 amino acid sequences identified for each patient
and for
each TCR chain. For each bar, starting from the x-axis, the bottom segment
represents the
most predominant CDR sequence. The next nine most predominant sequences are
stacked
above the bottom segment and are ordered by decreasing frequency going
upwards. The
remaining sequences are grouped together in the top segment. WT1, Wilms Tumor
1;
CDR3, complementarity determining region 3.
DESCRIPTION OF THE INVENTION
The terms "comprising", "comprises" and "comprised of' as used herein are
synonymous
with "including" or "includes"; or "containing" or "contains", and are
inclusive or open-ended
and do not exclude additional, non-recited members, elements or steps. The
terms
"comprising", "comprises" and "comprised of' also include the term "consisting
of".
T-cell receptor
During antigen processing, antigens are degraded inside cells and then carried
to the cell
surface by major histocompatibility complex (MHC) molecules. T-cells are able
to recognise
this peptide:MHC complex at the surface of the antigen presenting cell. There
are two
different classes of MHC molecules: MHC I and MHC II, each class delivers
peptides from
different cellular compartments to the cell surface.
A T cell receptor (TCR) is a molecule which can be found on the surface of T-
cells that is
responsible for recognizing antigens bound to MHC molecules. The naturally-
occurring TCR
heterodimer consists of an alpha (a) and beta (13) chain in around 95% of T-
cells, whereas
around 5% of T-cells have TCRs consisting of gamma (y) and delta (6) chains.
Engagement of a TCR with antigen and MHC results in activation of the T
lymphocyte on
which the TCR is expressed through a series of biochemical events mediated by
associated
enzymes, co-receptors, and specialized accessory molecules.
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Each chain of a natural TCR is a member of the immunoglobulin superfamily and
possesses
one N-terminal immunoglobulin OM-variable (V) domain, one Ig-constant (C)
domain, a
transmembrane/cell membrane-spanning region, and a short cytoplasmic tail at
the C-
terminal end.
The variable domain of both the TCR a chain and 13 chain have three
hypervariable or
complementarity determining regions (CDRs). A TCR a chain or 13 chain, for
example,
comprises a CDR1, a CDR2, and a CDR3 in amino to carboxy terminal order. In
general,
CDR3 is the main CDR responsible for recognizing processed antigen, although
CDR1 of
the alpha chain has also been shown to interact with the N-terminal part of
the antigenic
peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of
the peptide.
CDR2 is thought to recognize the MHC molecule.
A constant domain of a TCR may consist of short connecting sequences in which
a cysteine
residue forms a disulfide bond, making a link between the two chains.
An a chain of a TCR of the present invention may have a constant domain
encoded by a
TRAC gene. An example amino acid sequence of an a chain constant domain
encoded by a
TRAC gene is a shown below:
I QNPDPAVYQLRD SKS SDKSVCLF TDFD S QTNVS Q SKD SDVY I TDKTVLDMRSMDFK
SNSAVAWSNKSDFACANAFNNS I IPEDTFFP SPE S SCDVKLVEKSFETDTNLNFQNL
SVIGFRILLLKVAGFNLLMTLRLWS S
(SEQ ID NO: 76)
A TCR of the invention may comprise an a chain comprising the amino acid
sequence of
SEQ ID NO: 76 or a variant thereof having at least 75%, at least 80%, at least
85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity
thereto, preferably at least 75% sequence identity thereto.
.. A 13 chain of a TCR of the present invention may have a constant domain
encoded by a
TRBC1 or a TRBC2 gene. An example amino acid sequence of a 13 chain constant
domain
encoded by a TRBC1 gene is a shown below:
DLNKVFPPEVAVFEP SEAE I SHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVS T
DP QP LKEQPALND SRYCL S SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK
PVTQIVSAEAWGRADCGFT SVS YQQGVL SAT I LYE I LLGKATLYAVLVSALVLMAMV
KRKDF
(SEQ ID NO: 77)
An example amino acid sequence of a 13 chain constant domain encoded by a
TRBC2 gene
is a shown below:
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DLKNVFPPEVAVFEP SEAE I SHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVS T
DP QP LKEQPALND SRYCL S SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK
PVTQIVSAEAWGRADCGFT SE S YQQGVL SAT I LYE I LLGKATLYAVLVSALVLMAMV
KRKDSRG
(SEQ ID NO: 78)
A TCR of the invention may comprise a 13 chain comprising the amino acid
sequence of SEQ
ID NO: 77, SEQ ID NO: 78, or variants of SEQ ID NOs: 77 and 78 having at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least
98%, at least 99% sequence identity thereto, preferably at least 75% sequence
identity
thereto.
The TCR of the invention may have one or more additional cysteine residues in
each of the
a and 13 chains such that the TCR may comprise two or more disulphide bonds in
the
constant domains.
Mutations of TCR constant domains disclosed herein may be described based on a
numbering convention in which the first amino acid of each of SEQ ID NOs: 76-
78 is
assigned to be position 2.
The structure allows the TCR to associate with other molecules like CD3 which
possess
three distinct chains (y, 6, and c) in mammals and the -chain. These accessory
molecules
have negatively charged transmembrane regions and are vital to propagating the
signal from
the TCR into the cell. The CD3- and -chains, together with the TCR, form what
is known as
the T cell receptor complex.
The signal from the T cell complex is enhanced by simultaneous binding of the
MHC
molecules by a specific co-receptor. For helper T-cells, this co-receptor is
CD4 (specific for
class ll MHC); whereas for cytotoxic T-cells, this co-receptor is CD8
(specific for class I
MHC). The co-receptor allows prolonged engagement between the antigen
presenting cell
and the T cell and recruits essential molecules (e.g., LCK) inside the cell
involved in the
signalling of the activated T lymphocyte.
Accordingly, as used herein the term "T-cell receptor" (TCR) refers to
molecule capable of
recognising a peptide when presented by an MHC molecule. The molecule may be a
heterodimer of two chains a and 13 (or optionally y and 6) or it may be a
single chain TCR
construct. A TCR of the invention may be a soluble TCR, e.g. omitting or
altering one or
more constant domains. A TCR of the invention may comprise a constant domain.
The invention also provides an a chain or a 13 chain from such a T cell
receptor.
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The TCR of the invention may be a hybrid TCR comprising sequences derived from
more
than one species. For example, it has been found that murine TCRs are more
efficiently
expressed in human T-cells than human TCRs. The TCR may therefore comprise a
human
variable region and murine sequences within a constant region.
A disadvantage of this approach is that the murine constant sequences may
trigger an
immune response, leading to rejection of the transferred T-cells. However, the
conditioning
regimens used to prepare patients for adoptive T-cell therapy may result in
sufficient
immunosuppression to allow the engraftment of T-cells expressing murine
sequences.
In one embodiment, the TCR comprises one or more mutations to remove one or
more N-
glycosylation sites. Preferably, the N-glycosylation sites are in the TCR
constant domains.
Deletion of N-glycosylation sites in TCR constant domains is described in
Kuball, J et al.
(2009) J Exp Med 206: 463-75. In one embodiment, the one or more mutations are
substitutions of the amino acid N in an N-X-S/T motif with the amino acid Q.
For example,
the substitution may at one or more of the positions: TCR alpha constant gene
position 36,
90 or 109; and/or TCR beta constant gene postion 85.6. Preferably, the
substitution is at
position 36 of the TCR alpha constant gene.
Complementarity determining (CDR) regions
The portion of the TCR that establishes the majority of the contacts with the
antigenic
peptide bound to the major histocompatibility complex (MHC) is the
complementarity
determining region 3 (CDR3), which is unique for each T cell clone. The CDR3
region is
generated upon somatic rearrangement events occurring in the thymus and
involving non-
contiguous genes belonging to the variable (V), diversity (D, for 13 and 6
chains) and joining
(J) genes. Furthermore, random nucleotides inserted/deleted at the rearranging
loci of each
TCR chain gene greatly increase diversity of the highly variable CDR3
sequence. Thus, the
frequency of a specific CDR3 sequence in a biological sample indicates the
abundance of a
specific T cell population. The great diversity of the TCR repertoire in
healthy human beings
provides a wide range protection towards a variety of foreign antigens
presented by MHC
molecules on the surface of antigen presenting cells. In this regard, it is of
note that
theoretically up to 1015 different TCRs can be generated in the thymus.
.. T-cell receptor diversity is focused on CDR3 and this region is primarily
responsible for
antigen recognition.
The sequences of the CDR3 regions of the TCR of the invention may be selected
from those
set out in Table 1 below. A TCR may comprise CDRs that comprise or consist of
a CDR3a
and a CDR313 pair described below.
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The CDRs may, for example, comprise one, two, or three substitutions,
additions or
deletions from the given sequence, provided that the TCR retains the capacity
to bind a WT1
peptide when presented by an MHC molecule.
As used herein, the term "protein" includes single-chain polypeptide molecules
as well as
multiple-polypeptide complexes where individual constituent polypeptides are
linked by
covalent or non-covalent means. As used herein, the term "polypeptide" refers
to a polymer
in which the monomers are amino acids and are joined together through peptide
or
disulphide bonds.
Variants, derivatives, analogues, homologues and fragments
In addition to the specific proteins and polynucleotides mentioned herein, the
invention also
encompasses the use of variants, derivatives, analogues, homologues and
fragments
thereof.
In the context of the invention, a variant of any given sequence is a sequence
in which the
specific sequence of residues (whether amino acid or nucleic acid residues)
has been
modified in such a manner that the polypeptide or polynucleotide in question
substantially
retains at least one of its endogenous functions. A variant sequence can be
obtained by
addition, deletion, substitution, modification, replacement and/or variation
of at least one
residue present in the naturally-occurring protein.
A variant amino acid sequence of the invention referred to as having up to
three amino acid
substitutions, additions or deletions may have, for example, one, two or three
amino acid
substitutions, additions or deletions.
The term "derivative" as used herein, in relation to proteins or polypeptides
of the invention
includes any substitution of, variation of, modification of, replacement of,
deletion of and/or
addition of one (or more) amino acid residues from or to the sequence
providing that the
resultant protein or polypeptide substantially retains at least one of its
endogenous functions.
The term "analogue" as used herein, in relation to polypeptides or
polynucleotides includes
any mimetic, that is, a chemical compound that possesses at least one of the
endogenous
functions of the polypeptides or polynucleotides which it mimics.
Proteins used in the invention may also have deletions, insertions or
substitutions of amino
acid residues which produce a silent change and result in a functionally
equivalent protein.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity,
charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic
nature of the
residues as long as the endogenous function is retained. For example,
negatively charged
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amino acids include aspartic acid and glutamic acid; positively charged amino
acids include
lysine and arginine; and amino acids with uncharged polar head groups having
similar
hydrophilicity values include asparagine, glutamine, serine, threonine and
tyrosine.
A substitution may involve replacement of an amino acid for a similar amino
acid (a
conservative substitution). A similar amino acid is one which has a side chain
moiety with
related properties as grouped together, for example as shown below:
(i) basic side chains: lysine (K), arginine (R), histidine (H);
(ii) acidic side chains: aspartic acid (D) and glutamic acid (E);
(iii) uncharged polar side chains: asparagine (N), glutamine (Q), serine (S),
threonine
(T) and tyrosine (Y); or
(iv) non-polar side chains: glycine (G), alanine (A), valine (V), leucine (L),
isoleucine
(I), proline (P), phenylalanine (F), methionine (M), tryptophan (W) and
cysteine (C).
Any amino acid changes should maintain the capacity of the TCR to bind WT1
peptide
presented by MHC molecules.
Variant sequences may comprise amino acid substitutions, additions, deletions
and/or
insertions. The variation may be concentrated in one or more regions, such as
the constant
regions, the linker, or the framework regions of the a or 13 chains, or they
may be spread
throughout the TCR molecule.
Conservative substitutions, additions or deletions may be made, for example
according to
the Table below. Amino acids in the same block in the second column and
preferably in the
same line in the third column may be substituted for each other:
ALIPHATIC Non-polar G A P
ILV
Polar ¨ uncharged CSTM
NQ
Polar - charged D E
KR
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AROMATIC H F WY
The invention also encompasses homologous substitution (substitution and
replacement are
both used herein to mean the interchange of an existing amino acid residue,
with an
alternative residue), e.g. like-for-like substitution such as basic for basic,
acidic for acidic,
polar for polar etc. Non-homologous substitution may also occur e.g. from one
class of
residue to another or alternatively involving the inclusion of unnatural amino
acids, such as
ornithine.
The term "variant" as used herein may mean an entity having a certain homology
with the
wild type amino acid sequence or the wild type nucleotide sequence. The term
"homology"
can be equated with "identity".
A variant sequence may include an amino acid sequence which may be at least
50%, 55%,
65%, 75%, 85% or 90% identical, preferably at least 95%, at least 97%, or at
least 99%
identical to the subject sequence. Typically, the variants will comprise the
same active sites
etc. as the subject amino acid sequence. Although homology can also be
considered in
terms of similarity (i.e. amino acid residues having similar chemical
properties/functions), in
the context of the invention it is preferred to express homology in terms of
sequence identity.
A variant sequence may include a nucleotide sequence which may be at least
40%, 45%,
50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, at least
97%, or at
least 99% identical to the subject sequence. Although homology can also be
considered in
terms of similarity, in the context of the invention it is preferred to
express homology in terms
of sequence identity.
Preferably, reference to a sequence which has a percent identity to any one of
the SEQ ID
NOs detailed herein refers to a sequence which has the stated percent identity
over the
entire length of the SEQ ID NO referred to.
Identity comparisons can be conducted by eye or, more usually, with the aid of
readily
available sequence comparison programs. These commercially available computer
programs can calculate percentage homology or identity between two or more
sequences.
Percentage homology may be calculated over contiguous sequences, i.e. one
sequence is
aligned with the other sequence and each amino acid in one sequence is
directly compared
with the corresponding amino acid in the other sequence, one residue at a
time. This is
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called an "ungapped" alignment. Typically, such ungapped alignments are
performed only
over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into
consideration that,
for example, in an otherwise identical pair of sequences, one insertion or
deletion in the
nucleotide sequence may cause the following codons to be put out of alignment,
thus
potentially resulting in a large reduction in percent homology when a global
alignment is
performed. Consequently, most sequence comparison methods are designed to
produce
optimal alignments that take into consideration possible insertions and
deletions without
penalising unduly the overall homology score. This is achieved by inserting
"gaps" in the
sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that
occurs in
the alignment so that, for the same number of identical amino acids, a
sequence alignment
with as few gaps as possible, reflecting higher relatedness between the two
compared
sequences, will achieve a higher score than one with many gaps. "Affine gap
costs" are
typically used that charge a relatively high cost for the existence of a gap
and a smaller
penalty for each subsequent residue in the gap. This is the most commonly used
gap
scoring system. High gap penalties will of course produce optimised alignments
with fewer
gaps. Most alignment programs allow the gap penalties to be modified. However,
it is
preferred to use the default values when using such software for sequence
comparisons.
For example when using the GCG Wisconsin Bestfit package the default gap
penalty for
amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum percentage homology therefore firstly requires the
production of an
optimal alignment, taking into consideration gap penalties. A suitable
computer program for
carrying out such an alignment is the GCG Wisconsin Bestfit package
(University of
Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Res. 12: 387).
Examples of other
software that can perform sequence comparisons include, but are not limited
to, the BLAST
package (see Ausubel etal. (1999) ibid ¨ Ch. 18), FASTA (Atschul et al. (1990)
J. Mol. Biol.
403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are
available for offline and online searching (see Ausubel et al. (1999) ibid,
pages 7-58 to 7-60).
However, for some applications, it is preferred to use the GCG Bestfit
program. Another
tool, called BLAST 2 Sequences is also available for comparing protein and
nucleotide
sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett.
(1999)
177: 187-8).
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Although the final percentage homology can be measured in terms of identity,
the alignment
process itself is typically not based on an all-or-nothing pair comparison.
Instead, a scaled
similarity score matrix is generally used that assigns scores to each pairwise
comparison
based on chemical similarity or evolutionary distance. An example of such a
matrix
commonly used is the BLOSUM62 matrix ¨ the default matrix for the BLAST suite
of
programs. GCG Wisconsin programs generally use either the public default
values or a
custom symbol comparison table if supplied (see the user manual for further
details). For
some applications, it is preferred to use the public default values for the
GCG package, or in
the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to
calculate
percentage homology, preferably percentage sequence identity. The software
typically does
this as part of the sequence comparison and generates a numerical result.
"Fragments" are also variants and the term typically refers to a selected
region of the
polypeptide or polynucleotide that is of interest either functionally or, for
example, in an
assay. "Fragment" thus refers to an amino acid or nucleic acid sequence that
is a portion of
a full-length polypeptide or polynucleotide.
Such variants may be prepared using standard recombinant DNA techniques such
as site-
directed mutagenesis. Where insertions are to be made, synthetic DNA encoding
the
insertion together with 5' and 3' flanking regions corresponding to the
naturally-occurring
sequence either side of the insertion site may be made. The flanking regions
will contain
convenient restriction sites corresponding to sites in the naturally-occurring
sequence so that
the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA
ligated into
the cut. The DNA is then expressed in accordance with the invention to make
the encoded
protein. These methods are only illustrative of the numerous standard
techniques known in
the art for manipulation of DNA sequences and other known techniques may also
be used.
Major histocompatability complex (MHC) molecules
Typically, TCRs bind to peptides as part of peptide:MHC complex.
The MHC molecule may be an MHC class I or ll molecule. The complex may be on
the
surface of an antigen presenting cell, such as a dendritic cell or a B cell,
or any other cell,
including cancer cells, or it may be immobilised by, for example, coating on
to a bead or
plate.
The human leukocyte antigen system (HLA) is the name of the gene complex which
encodes major histocompatibility complex (MHC) in humans and includes HLA
class I
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antigens (A, B & C) and HLA class ll antigens (DP, DQ, & DR). HLA alleles A, B
and C
present peptides derived mainly from intracellular proteins, e.g. proteins
expressed within
the cell. This is of particular relevance since WT1 is an intracellular
protein.
During T-cell development in vivo, T-cells undergo a positive selection step
to ensure
.. recognition of self MHCs followed by a negative step to remove T-cells that
bind too strongly
to MHC which present self-antigens. As a consequence, certain T-cells and the
TCRs they
express will only recognise peptides presented by certain types of MHC
molecules - i.e.
those encoded by particular HLA alleles. This is known as HLA restriction.
One HLA allele of interest is HLA-A*0201, which is expressed in the vast
majority (>50%) of
the Caucasian population. Accordingly, TCRs which bind WT1 peptides presented
by MHC
encoded by HLA-A*0201 (i.e. are HLA-A*0201 restricted) are advantageous since
an
immunotherapy making use of such TCRs will be suitable for treating a large
proportion of
the Caucasian population.
Other HLA alleles of interest are HLA-B*38:01, HLA-C*03:03 and HLA-C*07:02.
.. Further alleles of interest are HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-
DRA
and HLA-DRB1. These are the six main MHC class II genes in humans.
In one embodiment, the TCR of the invention is HLA-A*0201-, HLA-A*0101-, HLA-
A*2402-,
H LA-A*0301-, H LA-B*3501- or HLA-B*0702-restricted.
A TCR of the present invention may be HLA-A*02:01-restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CILSTRVWAGSYQLTF (SEQ ID NO: 14) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino acid
sequence of CATGQATQETQYF (SEQ ID NO: 19) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CASGGGADGLTF (SEQ ID NO: 25) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino acid
sequence of CASGRGDTEAFF (SEQ ID NO: 30) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof having up to
three amino
acid substitutions, additions or deletions, and a CDR3[3 comprising the amino
acid sequence
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of CASSSGLAFYEQYF (SEQ ID NO: 98) or a variant thereof having up to three
amino acid
substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof having up to
three amino
acid substitutions, additions or deletions, and a CDR3[3 comprising the amino
acid sequence
of CASSQLSGRDSYEQYF (SEQ ID NO: 104) or a variant thereof having up to three
amino
acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino acid
sequence of CASSTLGGELFF (SEQ ID NO: 120) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino acid
sequence of CASSTLGGELFF (SEQ ID NO: 120) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof having
up
to three amino acid substitutions, additions or deletions, and a CDR3[3
comprising the amino
acid sequence of CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136) or a variant thereof
having up to three amino acid substitutions, additions or deletions, the TCR
is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof having
up
to three amino acid substitutions, additions or deletions, and a CDR3[3
comprising the amino
acid sequence of CATSWGLNEQYF (SEQ ID NO: 142) or a variant thereof having up
to
three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAATSRDDMRF (SEQ ID NO: 131) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino acid
sequence of CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136) or a variant thereof having
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up to three amino acid substitutions, additions or deletions, the TCR is HLA-
A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAATSRDDMRF (SEQ ID NO: 131) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CATSWGLNEQYF (SEQ ID NO: 142) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CALPDKVIF (SEQ ID NO: 148) or a variant thereof having up to three
amino
acid substitutions, additions or deletions, and a CDR38 comprising the amino
acid sequence
of CASSVSAGSTGELFF (SEQ ID NO: 158) or a variant thereof having up to three
amino
acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAGLYATNKLIF (SEQ ID NO: 153) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CASSVSAGSTGELFF (SEQ ID NO: 158) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof having up to
three amino
acid substitutions, additions or deletions, and a CDR38 comprising the amino
acid sequence
of CASSTLGGELFF (SEQ ID NO: 120) or a variant thereof having up to three amino
acid
substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CASSSGLAFYEQYF (SEQ ID NO: 98) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CASSQLSGRDSYEQYF (SEQ ID NO: 104) or a variant thereof having up
to
three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
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In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR36 comprising the
amino acid
sequence of CASSSGLAFYEQYF (SEQ ID NO: 98) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR36 comprising the
amino acid
sequence of CASSQLSGRDSYEQYF (SEQ ID NO: 104) or a variant thereof having up
to
three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01
restricted.
In one embodiment, a TCR of the present invention that is HLA-A*02:01
restricted binds to a
WT1 peptide comprising amino acid sequence LLAAILDFLLLQDPA (SEQ ID NO: 82) or
a
variant thereof having up to three amino acid substituions, additions or
deletions.
In one aspect, the invention provides a TCR which binds a Wilms tumour 1
protein (WT1)
peptide when presented by a major histocompatibility complex (MHC), wherein
the TCR
comprises a CDR3a comprising the amino acid sequence of CILSTRVWAGSYQLTF (SEQ
ID NO: 14) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, and a CDR36 comprising the amino acid sequence of CATGQATQETQYF
(SEQ
ID NO: 19) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1
peptide
comprises the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a
variant
thereof having up to three amino acid substituions, additions or deletions.
In one aspect, the invention provides a TCR which binds a Wilms tumour 1
protein (WT1)
peptide when presented by a major histocompatibility complex (MHC), wherein
the TCR
comprises a CDR3a comprising the amino acid sequence of CASGGGADGLTF (SEQ ID
NO: 25) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, and a CDR36 comprising the amino acid sequence of CASGRGDTEAFF (SEQ
ID
NO: 30) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1
peptide
comprises the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a
variant
thereof having up to three amino acid substituions, additions or deletions.
Another widely expressed HLA allele of interest is HLA-B*38:01. A TCR of the
invention
may be HLA- B*38:01 restricted.
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In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAMRTGGGADGLTF (SEQ ID NO: 3) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino acid
sequence of CASSEAGLSYEQYF (SEQ ID NO: 8) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-B*38:01
restricted.
In one aspect, the invention provides a TCR which binds a Wilms tumour 1
protein (WT1)
peptide when presented by a major histocompatibility complex (MHC), wherein
the TCR
comprises a CDR3a comprising the amino acid sequence of CAMRTGGGADGLTF (SEQ ID
NO: 3) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, and a CDR3[3 comprising the amino acid sequence of CASSEAGLSYEQYF
(SEQ
ID NO: 8) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, wherein the TCR is HLA-B*38:01 restricted, and wherein the WT1
peptide
comprises the amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or a
variant
thereof having up to three amino acid substituions, additions or deletions.
Another widely expressed HLA allele of interest is HLA-C*07:02. A TCR of the
invention
may be HLA- C*07:02 restricted.
In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CAMRTGGGADGLTF (SEQ ID NO: 3) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR3[3 comprising the
amino acid
sequence of CASSEAGLSYEQYF (SEQ ID NO: 8) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-C*07:02
restricted.
In one aspect, the invention provides a TCR which binds a Wilms tumour 1
protein (WT1)
peptide when presented by a major histocompatibility complex (MHC), wherein
the TCR
comprises a CDR3a comprising the amino acid sequence of CAMRTGGGADGLTF (SEQ ID
.. NO: 3) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, and a CDR3[3 comprising the amino acid sequence of CASSEAGLSYEQYF
(SEQ
ID NO: 8) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, wherein the TCR is HLA-C*07:02 restricted, and wherein the WT1
peptide
comprises the amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or a
variant
thereof having up to three amino acid substituions, additions or deletions.
Another widely expressed HLA allele of interest is HLA-C*03:03. A TCR of the
invention
may be HLA- C*03:03 restricted.
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In one aspect, where a TCR of the invention comprises a CDR3a comprising the
amino acid
sequence of CASGGGADGLTF (SEQ ID NO: 25) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR36 comprising the
amino acid
sequence of CASGRGDTEAFF (SEQ ID NO: 30) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, the TCR is HLA-C*03:03
restricted.
In one aspect, the invention provides a TCR which binds a Wilms tumour 1
protein (WT1)
peptide when presented by a major histocompatibility complex (MHC), wherein
the TCR
comprises a CDR3a comprising the amino acid sequence of CASGGGADGLTF (SEQ ID
NO: 25) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, and a CDR36 comprising the amino acid sequence of CASGRGDTEAFF (SEQ
ID
NO: 30) or a variant thereof having up to three amino acid substitutions,
additions or
deletions, wherein the TCR is HLA-C*03:03 restricted, and wherein the WT1
peptide
comprises the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a
variant
thereof having up to three amino acid substituions, additions or deletions.
In one embodiment, where a TCR of the invention binds to a WT1 peptide
comprising an
amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a variant thereof
having up
to three amino acid substitutions, additions or deletions, the TCR is HLA-
A*02:01 restricted.
In one embodiment, where a TCR of the invention binds to a WT1 peptide
comprising an
amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or a variant thereof
having
up to three amino acid substitutions, additions or deletions, the TCR is HLA-
B*38:01
restricted.
In one embodiment, where a TCR of the invention binds to a WT1 peptide
comprising an
amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or a variant thereof
having
up to three amino acid substitutions, additions or deletions, the TCR is HLA-
C*07:02
restricted.
In one embodiment, where a TCR of the invention binds to a WT1 peptide
comprising an
amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a variant thereof
having up
to three amino acid substitutions, additions or deletions, the TCR is HLA-
C*03:03 restricted.
Wilms tumor 1 (WT1) protein
Wilms tumor 1 (WT1) is an intracellular protein encoding a zinc finger
transcription factor that
plays an important role in cell growth and differentiation (Yang, L. et al.
Leukemia 21, 868-
876 (2007)). It is widely expressed on a variety of hematological and solid
tumors, while
showing limited expression on other tissues (gonads, uterus, kidney,
mesothelium,
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progenitor cells in different tissues). Recent evidence suggests that WT1
plays a role in
leukemogenesis and tumorigenesis.
WT1 has several isoforms, some of which result from alternative splicing of
mRNA
transcripts encoding WT1. The complete amino acid sequence of a WT1 isoform
was
previously published (Gessler, M. etal. Nature; 343(6260):774-778; (1990)).
This particular
isoform consists of 575 amino acids and includes a first 126 amino acids at
the N terminus
which are lacking in the exon 5+ and the KTS+ isoforms of WT1.
An example WT1 protein has the amino acid sequence set out in UniProt entry
J3KNN9.
Another example WT1 protein has the amino acid sequence set out below:
SRQRP HP GALRNP TACP LP HFP P S LP P T HSP T HP PRAGTAAQAP GP RRLLAAI LDF
LLLQDP
AS TCVP EPASQHTLRS GP GCLQQPEQQGVRDP GGIWAKLGAAEASAERLQGRRSRGASGSEP
QQMGSDVRDLNALLPAVP SLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAP
PP PP PP PP HSF IKQEP SWGGAEP HEEQCLSAF TVHF SGQF TGTAGACRYGPF GP PP P SQAS S
GQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTP SYGHTP SHHAAQFPNHSFKHEDPMGQ
QGSLGEQQYSVP PPVYGCHTP TDS CT GSQALLLRTP YS SDNLYQMTSQLECMTWNQMNLGAT
LKGVAAGS SS SVKWTEGQSNHSTGYESDNHTTP I LCGAQYRI HT HGVFRGIQDVRRVP GVAP
TLVRSASETSEKRPFMCAYP GCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQL
KRHQRRHT GVKP FQCKTCQRKF SRSDHLKT HTRT HT GKT SEKPF SCRWP S CQKKFARSDELV
RHHNMHQRNMTKLQLAL
(SEQ ID NO: 79)
WTI peptides
As used herein the term peptide refers to a plurality of amino acid residues
linked by peptide
bonds. As defined herein a peptide may consist of less than about 30, less
than about 25,
less than about 20, less than 19, less than 18, less than 17, less than 16,
less than 15, less
than 14, less than 13, less than 12, less than 11, less than 10, less than 9,
less than 8, less
than 7, less than 6, or less than 5 amino acid residues in length. Preferably,
a peptide is
about 5 to 20 amino acids in length, more preferably, a peptide is about 8 to
15 amino acid
residues in length.
The TCRs of the invention bind to a WT1 peptide when presented by an MHC. As
used
herein, the term WT1 peptide is understood to mean a peptide comprising an
amino acid
sequence derived from a WT1 protein.
For example, a WT1 peptide may comprise at least 5, at least 6, at least 7, at
least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, or at least 25 contiguous
amino acid residues of
a WT1 protein amino acid sequence.
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The WT1 peptide may comprise or consist of an amino acid sequence selected
from the
group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID
NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87) or variants thereof each having up
to
three amino acid substitutions, additions or deletions.
In some embodiments, for WT1 peptides which bind to MHC molecules encoded by
HLA-
A*0201 allele it may be preferred that the amino acids at position 2 of the
peptide (i.e. the
second amino acid from the N-terminus) are leucine or methionine, although
isoleucine,
valine, alanine and threonine may also be preferable. It may also be preferred
that the
amino acid at position 9 or 10 is valine, leucine or isoleucine, although
alanine, methionine
and threonine may also be preferable. The preferred MHC binding motifs of
other HLA
alleles are disclosed in Celis et al (Molecular Immunology, Vol. 31, 8,
December 1994,
pages 1423 to 1430).
Various uses of the WT1 peptides described herein are contemplated by the
invention. For
example, the WT1 peptides described herein may be administered to a subject,
e.g. a
human subject. Administration of the WT1 peptides of the invention may elicit
an immune
response against cells expressing or overexpressing WT1 protein, i.e. the WT1
peptides are
immunogenic WT1 peptides.
Thus in another aspect, the invention provides an isolated immunogenic WT1
peptide
comprising an amino acid sequence selected from the group consisting of
GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and
CMTWNQMNLGATLKG (SEQ ID NO: 87), and variants thereof each having up to three
amino acid substitutions, additions or deletions.
The WT1 peptides described herein, e.g. WT1 peptides comprising an amino acid
sequence
selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181),
LLAAILDFLLLQDPA (SEQ ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87), and
variants thereof each having up to three amino acid substitutions, additions
or deletions, may
be used to screen for and/or identify new TCR sequences which bind to WT1
cells. For
example, T2 cells may be pulsed with a WT1 peptide mentioned in the invention
and
incubated with a T-cell population isolated from a donor. In this approach,
expression of
cytokines, e.g. CD107a and IFNy, may be indicative of T-cells which recognise
WT1
peptides.
Accordingly, in one aspect, the invention provides a T-cell receptor (TCR),
which binds to a
Wilms tumour 1 protein (WT1) peptide when presented by a major
histocompatibility
complex (MHC), wherein the WT1 peptide comprises an amino acid sequence
selected from
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the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ
ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87), and variants thereof each
having
up to three amino acid substitutions, additions or deletions.
TCR sequences
We have determined the amino acid sequences for TCRs that bind to WT1 peptides
described herein. In particular, we have determined the amino acid sequences
of the TCR
CDRs, which are important for WT1 peptide recognition and binding.
In one aspect, the invention provides a TCR comprising a CDR3a comprising the
amino acid
sequence of CILSTRVWAGSYQLTF (SEQ ID NO: 14) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CATGQATQETQYF (SEQ ID NO: 19) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, which binds to a WT1 peptide
comprising
the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a variant
thereof
having up to three amino acid substituions, additions or deletions when
presented by an
MHC.
In one aspect, the invention provides a TCR comprising a CDR3a comprising the
amino acid
sequence of CASGGGADGLTF (SEQ ID NO: 25) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CASGRGDTEAFF (SEQ ID NO: 30) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, which binds to a WT1 peptide
comprising
the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a variant
thereof
having up to three amino acid substituions, additions or deletions when
presented by an
MHC.
In one aspect, the invention provides a TCR comprising a CDR3a comprising the
amino acid
sequence of CAMRTGGGADGLTF (SEQ ID NO: 3) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CASSEAGLSYEQYF (SEQ ID NO: 8) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, which binds to a WT1 peptide
comprising
the amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or a variant
thereof
having up to three amino acid substituions, additions or deletions when
presented by an
MHC.
In one aspect, the invention provides a TCR comprising a CDR3a comprising the
amino acid
sequence of CAVIGGTDSWGKLQF (SEQ ID NO: 36) or a variant thereof having up to
three
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amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CASSQEEGAVYGYTF (SEQ ID NO: 41) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, which binds to a WT1 peptide
comprising
the amino acid sequence of CMTWNQMNLGATLKG (SEQ ID NO: 87) or a variant
thereof
having up to three amino acid substituions, additions or deletions when
presented by an
MHC.
In one aspect, the invention provides a TCR comprising a CDR3a comprising the
amino acid
sequence of CAVIGGTDSWGKLQF (SEQ ID NO: 36) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, and a CDR38 comprising the
amino acid
sequence of CATSREGLAADTQYF (SEQ ID NO: 52) or a variant thereof having up to
three
amino acid substitutions, additions or deletions, which binds to a WT1 peptide
comprising
the amino acid sequence of CMTWNQMNLGATLKG (SEQ ID NO: 87) or a variant
thereof
having up to three amino acid substituions, additions or deletions when
presented by an
MHC.
In one aspect, the invention provides a TCR comprising a CDR3a comprising the
amino acid
sequence of CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof having up
to
three amino acid substitutions, additions or deletions, and a CDR38 comprising
the amino
acid sequence of CATSREGLAADTQYF (SEQ ID NO: 52) or a variant thereof having
up to
three amino acid substitutions, additions or deletions, which binds to a WT1
peptide
comprising the amino acid sequence of CMTWNQMNLGATLKG (SEQ ID NO: 87) or a
variant thereof having up to three amino acid substituions, additions or
deletions when
presented by an MHC.
In one aspect, the invention provides a TCR comprising a CDR3a comprising the
amino acid
sequence of CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof having up
to
three amino acid substitutions, additions or deletions, and a CDR38 comprising
the amino
acid sequence of CASSQEEGAVYGYTF (SEQ ID NO: 41) or a variant thereof having
up to
three amino acid substitutions, additions or deletions, which binds to a WT1
peptide
comprising the amino acid sequence of CMTWNQMNLGATLKG (SEQ ID NO: 87) or a
variant thereof having up to three amino acid substituions, additions or
deletions when
presented by an MHC.
Example TCR amino acid sequences of the present invention are provided in
Table 1.
Table 1
Donor: HD12
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Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a TSDQSYG SEQ ID NO: 1
CDR2a QGSYDEQN SEQ ID NO: 2
CDR3a CAMRTGGGADGLTF SEQ ID NO: 3
Variable MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLD SEQ ID NO: 4
CTYDTSDQSYGLFWYKQP SSGEMI FL IYQGSYDEQNATEGRY
SLNFQKARKSANLVI SAS QLGD SAMYFCAMRTGGGADGLTFG
KGTHLI IQPY
Full ¨ with MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLD SEQ ID NO: 5
TRAC constant CTYDTSDQSYGLFWYKQP SSGEMI FL IYQGSYDEQNATEGRY
domain SLNFQKARKSANLVI SAS QLGD SAMYFCAMRTGGGADGLTFG
KGTHLI IQPY IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTN
VSQSKDSDVY ITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNS I IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSV
IGFRILLLKVAGFNLLMTLRLWSS
Full ¨ with MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLD SEQ ID NO: 194
TRAC constant CTYDTSDQSYGLFWYKQP SSGEMI FL IYQGSYDEQNATEGRY
domain SLNFQKARKSANLVI SAS QLGD SAMYFCAMRTGGGADGLTFG
KGTHLI IQPY IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTN
VSQSKDSDVY ITDKCVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNS I IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSV
IGFRILLLKVAGFNLLMTLRLWSS
Beta (13) CDR113 SNHLY SEQ ID NO: 6
CDR2I3 FYNNEI SEQ ID NO: 7
CDR3I3 CASSEAGLSYEQYF SEQ ID NO: 8
Variable MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRC SEQ ID NO: 9
VP I SNHLYFYWYRQ ILGQKVEFLVSFYNNE I SEKSE IFDDQF
SVERPDGSNFTLKI RS TKLEDSAMYFCASSEAGLSYEQYFGP
GTRLTVTE
Full ¨ with MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRC SEQ ID NO: 10
TRBC1 VP I SNHLYFYWYRQ ILGQKVEFLVSFYNNE I SEKSE IFDDQF
constant SVERPDGSNFTLKI RS TKLEDSAMYFCASSEAGLSYEQYFGP
domain GTRLTVTEDLNKVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTSVS YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDF
Full ¨ with MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRC SEQ ID NO: 11
TRBC2 VP I SNHLYFYWYRQ ILGQKVEFLVSFYNNE I SEKSE IFDDQF
constant SVERPDGSNFTLKI RS TKLEDSAMYFCASSEAGLSYEQYFGP
domain GTRLTVTEDLKNVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTS ES YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDS RG
Full ¨ with MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRC SEQ ID NO: 195
TRBC2 VP I SNHLYFYWYRQ ILGQKVEFLVSFYNNE I SEKSE IFDDQF
constant SVERPDGSNFTLKI RS TKLEDSAMYFCASSEAGLSYEQYFGP
domain GTRLTVTEDLKNVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTS ES YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDS RG
Donor: HD13
Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a TISGTDY SEQ ID NO: 12
CDR2a GLTSN SEQ ID NO: 13
CDR3a CI LS TRVWAGSYQLTF SEQ ID NO: 14
Variable MKLVTS ITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHS SEQ ID NO:
15
TI SGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAED
RKSS TL ILHRATLRDAAVYYCI LS TRVWAGSYQLTFGKGTKL
SVIPN
53
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Full ¨ with MKLVTS I TVLLS LGIMGDAKTTQPNSME SNEEEPVHLP CNHS SEQ ID NO: 16
TRAC constant T I SGTDY I HWYRQLP S QGPEYVIHGLTSNVNNRMAS LAIAED
domain RKS S TL ILHRATLRDAAVYYC I LS TRVWAGSYQLTFGKGTKL
SVIPNI QNPDPAVYQLRD SKS S DKSVCLFTDFDS QTNVSQSK
DS DVY I TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS
I I PEDTFFP SPE S S CDVKLVEKSFETDTNLNFQNLSVI GFRI
LLLKVAGFNLLMTLRLWS S
Full ¨ with MKLVTS I TVLLS LGIMGDAKTTQPNSME SNEEEPVHLP CNHS SEQ ID NO: 196
TRAC constant T I SGTDY I HWYRQLP S QGPEYVIHGLTSNVNNRMAS LAIAED
domain RKS S TL ILHRATLRDAAVYYC I LS TRVWAGSYQLTFGKGTKL
SVIPNI QNPDPAVYQLRD SKS S DKSVCLFTDFDS QTNVSQSK
DS DVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS
I I PEDTFFP SPE S S CDVKLVEKSFETDTNLNFQNLSVI GFRI
LLLKVAGFNLLMTLRLWS S
Beta (0) CDR1I3 KGHDR SEQ ID NO: 17
CDR2I3 SFDVKD SEQ ID NO: 18
CDR3I3 CATGQATQETQYF SEQ ID NO: 19
Variable MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLEC SEQ ID NO: 20
SQTKGHDRMYWYRQDP GLGLRL I YYSFDVKD INKGE I S DGYS
VSRQAQAKFS LS LE SAIPNQTALYFCATGQATQETQYFGP GT
RLLVLE
Full ¨ with MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLEC SEQ ID NO: 21
TRBC1 SQTKGHDRMYWYRQDP GLGLRL I YYSFDVKD INKGE I S DGYS
constant VSRQAQAKFS LS LE SAIPNQTALYFCATGQATQETQYFGP GT
domain RLLVLEDLNKVFPP EVAVFEP S EAE I SHTQKATLVCLATGFF
PDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SR
LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ IV
SAEAWGRADCGF TSVS YQQGVLSAT I LYE I LLGKATLYAVLV
SALVLMAMVKRKDF
Full ¨ with MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLEC SEQ ID NO: 22
TRBC2 SQTKGHDRMYWYRQDP GLGLRL I YYSFDVKD INKGE I S DGYS
constant VSRQAQAKFS LS LE SAIPNQTALYFCATGQATQETQYFGP GT
domain RLLVLEDLKNVFPP EVAVFEP S EAE I SHTQKATLVCLATGFY
PDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SR
LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ IV
SAEAWGRADCGF TS ES YQQGVLSAT I LYE I LLGKATLYAVLV
SALVLMAMVKRKDS RG
Full ¨ with MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLEC SEQ ID NO: 197
TRBC2 SQTKGHDRMYWYRQDP GLGLRL I YYSFDVKD INKGE I S DGYS
constant VSRQAQAKFS LS LE SAIPNQTALYFCATGQATQETQYFGP GT
domain RLLVLEDLKNVFPP EVAVFEP S EAE I SHTQKATLVCLATGFY
PDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLS SR
LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ IV
SAEAWGRADCGF TS ES YQQGVLSAT I LYE I LLGKATLYAVLV
SALVLMAMVKRKDS RG
Donor: HD14
Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a NSAFQY SEQ ID NO: 23
(with CDR2a TY S S GN SEQ ID NO: 24
TRAV12- CDR3a CASGGGADGLTF SEQ ID NO: 25
3*01) Variable mtlKsLRyLLviLwLQLswvwsQQKEvEupGpLsvpEGAivs SEQ ID NO: 26
LNCTYSNSAFQYFMWYRQYSRKGPELLMYTYS SGNKEDGRFT
AQVDKS SKY I SLF I RD SQP S DSATYLCASGGGADGLTFGKGT
HLIIQPY
Full ¨ with MMKS LRVLLVILwLQLSWVWSQQKEVEQDP GP LSVP EGAIVS SEQ ID NO: 27
TRAC constant LNCTYSNSAFQYFMWYRQYSRKGPELLMYTYS SGNKEDGRFT
domain AQVDKS SKY I SLF I RD SQP S DSATYLCASGGGADGLTFGKGT
HL I I QP Y I QNPDPAVYQLRD SKS S DKSVCLFTDFDS QTNVSQ
SKDS DVY I TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFN
NS I I PEDTFFP SPE S S CDVKLVEKSFETDTNLNFQNLSVI GF
RI LLLKVAGFNLLMTLRLWS S
Alpha (a) CDR1a DRGS QS SEQ ID NO: 182
(with CDR2a I Y SNGD SEQ ID NO: 183
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TRAV12- CDR3a CASGGGADGLTF SEQ ID
NO: 25
2*01) Variable mKsLRyLLviLwLQLswvwsQuEvEQNsGpLsvpEGAIAsL SEQ ID NO: 185
NCTYSDRGSQSFFWYRQYSGKSPELIMF TY SNGDKEDGRFTA
QLNKAS QYVS LL I RDS QP SD SATYLCAS GGGADGLTFGKGTH
LI IQPY
Full ¨ with MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASL SEQ ID NO: 186
TRAC constant NCTYSDRGSQSFFWYRQYSGKSPELIMF TY SNGDKEDGRFTA
domain QLNKAS QYVS LL I RDS QP SD SATYLCAS GGGADGLTFGKGTH
LI IQPY IQNP DPAVYQLRDSKS SDKSVCLFTDFDSQTNVS QS
KDSDVY ITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNN
SI IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSVIGFR
I LLLKVAGFNLLMTLRLWS S
Alpha (a) CDR1a DRGS QS SEQ
ID NO: 182
(with CDR2a TY SNGD SEQ
ID NO: 183
TRAV12- CDR3a CASGGGADGLTF SEQ ID
NO: 25
2*02) Variable mmKsLRyLLviLwLQLswvwsQQKEvEQNsGpLsvpEGAIAs SEQ ID NO:
190
LNCTYSDRGS QSFFWYRQYSGKSP EL IMS I YSNGDKEDGRFT
AQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGT
HLIIQPY
Full ¨ with MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIAS SEQ ID NO: 191
TRAC constant LNCTYSDRGS QSFFWYRQYSGKSP EL IMS I YSNGDKEDGRFT
domain AQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGT
HL I I QP Y I QNPDPAVYQLRDSKSSDKSVCLFTDFDS QTNVSQ
SKDS DVY I TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFN
NS I I PEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVI GF
RI LLLKVAGFNLLMTLRLWS S
Alpha (a) Full ¨ with MMKSLRVLLVIDwDQLSWVWSQQKEVEQDP GP LSVP EGAIVS SEQ ID NO:
198
(TRAV12- TRAC constant LNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFT
3*01 WT) domain AQVDKS SKY I SLF I RDSQP SDSATYLCASGGGADGLTFGKGT
HL I I QP Y I QNPDPAVYQLRDSKSSDKSVCLFTDFDS QTNVSQ
SKDS DVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFN
NS I I PEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVI GF
RI LLLKVAGFNLLMTLRLWS S
Alpha (a) Full ¨ with MKSLRVLLVI DwDQLSWVWS QQKEVEQNSGPLSVPEGAIASL SEQ ID NO:
199
(TRAV12- TRAC constant NCTYSDRGSQSFFWYRQYSGKSPELIMF TY SNGDKEDGRFTA
2*01 WT) domain QLNKAS QYVS LL I RDS QP SD SATYLCAS GGGADGLTFGKGTH
LI IQPY IQNP DPAVYQLRDSKS SDKSVCLFTDFDSQTNVS QS
KDSDVY ITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNN
SI IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSVIGFR
I LLLKVAGFNLLMTLRLWS S
Alpha (a) Full ¨ with MKSLRVLLVI DwDQLSWVWS QQKEVEQNSGPLSVPEGAIASL SEQ ID NO:
200
(TRAV12- TRAC constant NCTYSDRGSQSFFWYRQYSGKSPELIMF TY SNGDKEDGRFTA
2*01 mut) domain QLNKAS QYVS LL I RDS QP SD SATYLCAS GGGADGLTFGKGTH
LI IQPY IQNP DPAVYQLRDSKS SDKSVCLFTDFDSQTQVS QS
KDSDVY ITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNN
SI IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSVIGFR
I LLLKVAGFNLLMTLRLWS S
Alpha (a) Full ¨ with MMKSLRVLLVIDwDQLSWVWSQQKEVEQNSGPLSVPEGAIAS SEQ ID NO:
201
(TRAV12- TRAC constant LNCTYSDRGS QSFFWYRQYSGKSP EL IMS I YSNGDKEDGRFT
2*02 WT) domain AQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGT
HL I I QP Y I QNPDPAVYQLRDSKSSDKSVCLFTDFDS QTNVSQ
SKDS DVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFN
NS I I PEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVI GF
RI LLLKVAGFNLLMTLRLWS S
Alpha (a) Full ¨ with MMKSLRVLLVIDwDQLSWVWSQQKEVEQNSGPLSVPEGAIAS SEQ ID NO:
202
(TRAV12- TRAC constant LNCTYSDRGS QSFFWYRQYSGKSP EL IMS I YSNGDKEDGRFT
2*02 mut) domain AQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGT
HL I I QP Y I QNPDPAVYQLRDSKSSDKSVCLFTDFDS QTQVSQ
SKDS DVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFN
NS I I PEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVI GF
RI LLLKVAGFNLLMTLRLWS S
Beta (13) CDR1I3 SGDLS SEQ ID
NO: 28
CDR2I3 YYNGEE SEQ ID
NO: 29
CDR3I3 CASGRGDTEAFF SEQ ID
NO: 30
Variable mGFRLLccvAFcLLGAGPvDsGvTQTpKHLITATGQRvTLRc SEQ ID NO: 31
SP RSGDLSVYWYQQSLDQGLQFLI QYYNGEERAKGNILERFS
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AQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTR
LTVVE
Full ¨ with MGFRLLCCVAFCLLGAGpVDSGVTQTpKHLITATGQRvTLRC SEQ ID NO: 32
TRBC1 SPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFS
constant AQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTR
domain LTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFP
DHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVS
ALVLMAMVKRKDF
Full ¨ with MGFRLLCCVAFCLLGAGPVDSGVTQTpKHLITATGQRVTLRC SEQ ID NO: 33
TRBC2 SPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFS
constant AQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTR
domain LTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYP
DHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVS
ALVLMAMVKRKDSRG
Beta (0) ( Full ¨ with MGFRLLCCVAFCLLGAGPVDSGVTQTpKHLITATGQRVTLRC SEQ ID NO:
203
TRAV12- TRBC2 SPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFS
3*01 WT) constant AQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTR
domain LTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYP
DHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVS
ALVLMAMVKRKDSRG
Donor: HD15
Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a DRGSQS SEQ ID NO: 34
S4 CDR2a IYSNGD SEQ ID NO: 35
populatio CDR3a CAVIGGTDSWGKLQF SEQ ID NO: 36
Variable mKsLRyLLviLwLQLswvwsQuEvEQNsGpLsvpEGAIAsL SEQ ID NO: 37
NCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTA
QLNKASQYVSLLIRDSQPSDSATYLCAVIGGTDSWGKLQFGA
GTQVVVTPD
Full ¨ with MKSLRVLLviLwLQLsWVWSQQKEVEQNSGPLSVpEGAIASL SEQ ID NO: 38
TRAC constant NCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTA
domain QLNKASQYVSLLIRDSQPSDSATYLCAVIGGTDSWGKLQFGA
GTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNV
SQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANA
FNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVI
GFRILLLKVAGFNLLMTLRLWSS
Full ¨ with MKSLRVLLviLwLQLsWVWSQQKEVEQNSGPLSVpEGAIASL SEQ ID NO: 214
TRAC constant NCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTA
domain QLNKASQYVSLLIRDSQPSDSATYLCAVIGGTDSWGKLQFGA
GTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNV
SQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANA
FNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVI
GFRILLLKVAGFNLLMTLRLWSS
Beta (0) CDR10 LGHNA SEQ ID NO: 39
S4 CDR20 YSLEER SEQ ID NO: 40
populatio CDR30 CASSQEEGAVYGYTF SEQ ID NO: 41
Variable mGcRLLccAvLcLLGAGELvPmErGvTupRELvmGmTNKKs SEQ ID NO: 42
LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPS
RFSPECPNSSHLFLHLHTLQPEDSALYLCASSQEEGAVYGYT
FGSGTRLTVVE
Full ¨ with MGCRLLCCAVLCLLGAGELVpMETGvTQTpRHLvmGmTNKKS SEQ ID NO: 43
TRBC1 LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPS
constant RFSPECPNSSHLFLHLHTLQPEDSALYLCASSQEEGAVYGYT
domain FGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCL
ATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY
CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATL
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YAVLVSALVLMAMVKRKDF
Full ¨ with MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKS SEQ ID NO: 44
TRBC2 LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPS
constant RF SP ECPNS S HLFLHLHTLQPEDSALYLCAS S QEEGAVYGYT
domain FGSGTRLTVVEDLKNVFPPEVAVFEP SEAE I S HTQKATLVCL
ATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY
CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADCGFT SE SYQQGVLSAT ILYE ILLGKATL
YAVLVSALVLMAMVKRKDSRG
Full ¨ with MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKS SEQ ID NO: 215
TRBC2 LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPS
constant RF SP ECPNS S HLFLHLHTLQPEDSALYLCAS S QEEGAVYGYT
domain FGSGTRLTVVEDLKNVFPPEVAVFEP SEAE I S HTQKATLVCL
ATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRY
CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADCGFT SE SYQQGVLSAT ILYE ILLGKATL
YAVLVSALVLMAMVKRKDSRG
Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a NSAS QS SEQ ID
NO: 45
S1 IFNg CDR2a VYSSGN
SEQ ID NO: 46
enriched CDR3a CVVPRGLSTDSWGKLQF SEQ ID
NO: 47
populatio Variable misLRyLLviLwLQLswvwsQRKEvEQDpGpFNypEGATvAF SEQ ID NO: 48
n NCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQ
LNRASQY I SLLI RD SKLSDSATYLCVVP RGLS TD SWGKLQFG
AGTQVVVTPD
Full ¨ with misLRyLLviLwLQLswvwsQRKEvEQDpGpFNypEGATvAF SEQ ID NO: 49
TRAC constant NCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQ
domain LNRASQY I SLLI RD SKLSDSATYLCVVP RGLS TD SWGKLQFG
AGTQVVVTPD I QNP DPAVYQLRDSKS SDKSVCLFTDFDSQTN
VS QSKD SDVY I TDKTVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNS I IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSV
IGFRILLLKVAGFNLLMTLRLWSS
Full ¨ with misLRyLLviLwLQLswvwsQRKEvEQDpGpFNypEGATvAF SEQ ID NO: 216
TRAC constant NCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQ
domain LNRASQY I SLLI RD SKLSDSATYLCVVP RGLS TD SWGKLQFG
AGTQVVVTPD I QNP DPAVYQLRDSKS SDKSVCLFTDFDSQTN
VS QSKD SDVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNS I IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSV
IGFRILLLKVAGFNLLMTLRLWSS
Beta (13) CDR1I3 LNHNV SEQ ID
NO: 50
S1 IFNg CDR2I3 YYDKDF
SEQ ID NO: 51
enriched CDR3I3 CATS REGLAADTQYF SEQ ID
NO: 52
populatio Variable mGPGLLHwmALcLLGTGHGDAmviQNpRyQvTQFGKpvTLsc SEQ ID NO: 53
n SQTLNHNVMYWYQQKS SQAPKLLFHYYDKDFNNEADTPDNFQ
SRRPNTSFCFLD IRSPGLGDAAMYLCATSREGLAADTQYFGP
GTRLTVLE
Full ¨ with MGPGLLHWMALCLLGTGHGDAMVI QNPRYQVTQFGKPVTLSC SEQ ID NO: 54
TRBC1 SQTLNHNVMYWYQQKS SQAPKLLFHYYDKDFNNEADTPDNFQ
constant SRRPNTSFCFLD IRSPGLGDAAMYLCATSREGLAADTQYFGP
domain GTRLTVLEDLNKVFPP EVAVFEP S EAE I SHTQKATLVCLATG
FFPD HVEL SWWVNGKEVHSGVS TDP QP LKE QPALND SRYC LS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGF TSVS YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDF
Full ¨ with MGPGLLHWMALCLLGTGHGDAMVI QNPRYQVTQFGKPVTLSC SEQ ID NO: 55
TRBC2 SQTLNHNVMYWYQQKS SQAPKLLFHYYDKDFNNEADTPDNFQ
constant SRRPNTSFCFLD IRSPGLGDAAMYLCATSREGLAADTQYFGP
domain GTRLTVLEDLKNVFPP EVAVFEP S EAE I SHTQKATLVCLATG
FYPD HVEL SWWVNGKEVHSGVS TDP QP LKE QPALND SRYC LS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGF TS ES YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDS RG
Full ¨ with MGPGLLHWMALCLLGTGHGDAMVI QNPRYQVTQFGKPVTLSC SEQ ID NO: 217
TRBC2 SQTLNHNVMYWYQQKS SQAPKLLFHYYDKDFNNEADTPDNFQ
constant SRRPNTSFCFLD IRSPGLGDAAMYLCATSREGLAADTQYFGP
domain GTRLTVLEDLKNVFPP EVAVFEP S EAE I SHTQKATLVCLATG
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FYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTS ES YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDS RG
Patient 1
Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a VSNAYN SEQ ID NO: 91
direct CDR2a GSKP SEQ ID NO: 92
sequenci CDR3a CAAPNDYKLSF SEQ ID NO: 93
ng upon Variable mALQsTLGAvwLGLLLNsLwKvAEsKDQvFQpsTvAssEGAv SEQ ID NO:
94
sorting VEIFCNHSVSNAYNFFWYLHFPGCAPRLLVKGSKPSQQGRYN
MTYERF SS SLLI LQVREADAAVYYCAAPNDYKLSFGAGTTVT
VRAN
Full ¨ with MALQSTLGAVWLGLLLNSLWKVAESKDQVFQP STVASSEGAV SEQ ID NO: 95
TRAC constant VEIFCNHSVSNAYNFFWYLHFPGCAPRLLVKGSKPSQQGRYN
domain MTYERF SS SLLI LQVREADAAVYYCAAPNDYKLSFGAGTTVT
VRANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKD
SDVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS I
IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
Beta (13) 1 CDR1I3 SEHNR SEQ ID
NO: 96
direct CDR2I3 FQNEAQ SEQ ID NO: 97
sequenci CDR3I3 CASS SGLAFYEQYF SEQ ID NO: 98
ng upon Variable mGTsLLcwmALcLLGADHADTGvsQNpRHKITKRGQNvTFRc SEQ ID NO:
99
sorting DP I SEHNRLYWYRQTLGQGP EFLTYFQNEAQLEKSRLLSDRF
SAERPKGSFSTLEIQRTEQGDSAMYLCASSSGLAFYEQYFGP
GTRLTVTE
Full ¨ with MGTSLLCWMALCLLGADHADTGVS QNPRHK I TKRGQNVTFRC SEQ ID NO: 100
TRBC1 DP I SEHNRLYWYRQTLGQGP EFLTYFQNEAQLEKSRLLSDRF
constant SAERPKGSFSTLEIQRTEQGDSAMYLCASSSGLAFYEQYFGP
domain GTRLTVTEDLNKVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTSVS YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDF
Full ¨ with MGTSLLCWMALCLLGADHADTGVS QNPRHK I TKRGQNVTFRC SEQ ID NO: 101
TRBC2 DP I SEHNRLYWYRQTLGQGP EFLTYFQNEAQLEKSRLLSDRF
constant SAERPKGSFSTLEIQRTEQGDSAMYLCASSSGLAFYEQYFGP
domain GTRLTVTEDLKNVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTS ES YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDS RG
Beta (13) 2 CDR1I3 SGHDN SEQ ID NO: 102
direct CDR2I3 FVKESK SEQ ID NO: 103
sequenci CDR3I3 CASS QLSGRDSYEQYF SEQ ID NO: 104
ng upon Variable MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRC SEQ ID NO:
105
sorting DP I S GHDNLYWYRRVMGKEI KFLLHFVKESKQDESGMPNNRF
LAERTGGTYSTLKVQPAELEDSGVYFCASSQLSGRDSYEQYF
GP GTRLTVTE
Full ¨ with MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRC SEQ ID NO: 106
TRBC1 DP I S GHDNLYWYRRVMGKEI KFLLHFVKESKQDESGMPNNRF
constant LAERTGGTYSTLKVQPAELEDSGVYFCASSQLSGRDSYEQYF
domain GP GTRLTVTEDLNKVFPP EVAVFEP SEAEI SHTQKATLVCLA
TGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC
LS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV
TQ IVSAEAWGRADCGFTSVS YQQGVLSAT I LYEI LLGKATLY
AVLVSALVLMAMVKRKDF
Full ¨ with MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRC SEQ ID NO: 107
TRBC2 DP I S GHDNLYWYRRVMGKEI KFLLHFVKESKQDESGMPNNRF
constant LAERTGGTYSTLKVQPAELEDSGVYFCASSQLSGRDSYEQYF
domain GP GTRLTVTEDLKNVFPP EVAVFEP SEAEI SHTQKATLVCLA
TGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC
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LS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV
TQ IVSAEAWGRADCGFTSES YQQGVLSAT I LYEI LLGKATLY
AVLVSALVLMAMVKRKDS RG
Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a VSGNPY SEQ ID NO: 108
1 CDR2a Y I TGDNLV SEQ ID NO: 109
growing CDR3a CAVRDGGATNKL I F SEQ ID NO: 110
colony Variable MASAP I SMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKC SEQ ID NO:
111
TY SVSGNP YLFWYVQYPNRGLQFLLKY I TGDNLVKGSYGFEA
EFNKSQTSFHLKKP SALVSDSALYFCAVRDGGATNKLIFGTG
TLLAVQPN
Full ¨ with MASAP I SMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKC SEQ ID NO: 112
TRAC constant TY SVSGNP YLFWYVQYPNRGLQFLLKY I TGDNLVKGSYGFEA
domain EFNKSQTSFHLKKP SALVSDSALYFCAVRDGGATNKLIFGTG
TLLAVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS
QSKDSDVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAF
NNS I IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSVIG
FRI LLLKVAGFNLLMTLRLWS S
Alpha (a) CDR1a NIATNDY SEQ ID NO: 113
2 CDR2a GYKTK SEQ ID NO: 114
growing CDR3a CLVGGYTGGFKT IF SEQ ID NO: 115
colony Variable MRQVARVIVFLTLSTLSLAKTTQP I SMDSYEGQEVNI TCSHN SEQ ID NO:
116
NIATNDY I TWYQQFP S QGPRF I IQGYKTKVTNEVASLF IPAD
RKSSTLSLPRVSLSDTAVYYCLVGGYTGGFKT IFGAGTRLFV
KAN
Full ¨ with MRQVARVIVFLTLSTLSLAKTTQP I SMDSYEGQEVNI TCSHN SEQ ID NO: 117
TRAC constant NIATNDY I TWYQQFP S QGPRF I IQGYKTKVTNEVASLF IPAD
domain RKSSTLSLPRVSLSDTAVYYCLVGGYTGGFKT IFGAGTRLFV
KANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS
DVY I TDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS II
PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILL
LKVAGFNLLMTLRLWSS
Beta() CDR1I3 MNHEY SEQ ID NO: 118
growing CDR2I3 SMNVEV SEQ ID NO: 119
colony CDR3I3 CASS TLGGELFF SEQ ID NO: 120
Variable MGPQLLGYVVLCLLGAGP LEAQVTQNPRYL I TVTGKKLTVTC SEQ ID NO:
121
SQNMNHEYMSWYRQDP GLGLRQ I YYSMNVEVTDKGDVP EGYK
VSRKEKRNFP LI LESP SPNQTSLYFCASSTLGGELFFGEGSR
LTVLE
Full ¨ with MGPQLLGYVVLCLLGAGP LEAQVTQNPRYL I TVTGKKLTVTC SEQ ID NO: 122
TRBC1 SQNMNHEYMSWYRQDP GLGLRQ I YYSMNVEVTDKGDVP EGYK
constant VSRKEKRNFP LI LESP SPNQTSLYFCASSTLGGELFFGEGSR
domain LTVLEDLNKVFPPEVAVFEP SEAE I S HTQKATLVCLATGFFP
DHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSVSYQQGVLSAT ILYEILLGKATLYAVLVS
ALVLMAMVKRKDF
Full ¨ with MGPQLLGYVVLCLLGAGP LEAQVTQNPRYL I TVTGKKLTVTC SEQ ID NO: 123
TRBC2 SQNMNHEYMSWYRQDP GLGLRQ I YYSMNVEVTDKGDVP EGYK
constant VSRKEKRNFP LI LESP SPNQTSLYFCASSTLGGELFFGEGSR
domain LTVLEDLKNVFPPEVAVFEP SEAE I S HTQKATLVCLATGFYP
DHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSESYQQGVLSAT ILYEILLGKATLYAVLVS
ALVLMAMVKRKDSRG
Patient 2
Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a SSVSVY SEQ ID NO: 124
1 CDR2a YLSGSTLV SEQ ID NO: 125
CDR3a CAVTLLS I EP SAGGYQKVTF SEQ ID NO: 126
Variable MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVELRC SEQ ID NO:
127
NY SS SVSVYLFWYVQYPNQGLQLLLKYLSGSTLVES INGFEA
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EFNKSQTSFHLRKP SVHI SDTAEYFCAVTLLS IEPSAGGYQK
VTFGIGTKLQVIPN
Full ¨ with MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVELRC SEQ ID NO: 128
TRAC constant NY SS SVSVYLFWYVQYPNQGLQLLLKYLSGSTLVES INGFEA
domain EFNKSQTSFHLRKP SVHI SDTAEYFCAVTLLS IEPSAGGYQK
VTFGIGTKLQVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFD
SQTNVSQSKDSDVY I TDKCVLDMRSMDFKSNSAVAWSNKSDF
ACANAFNNS I IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQ
NLSVIGFRILLLKVAGFNLLMTLRLWSS
Alpha (a) CDR1a DSASNY SEQ
ID NO: 129
2 CDR2a IRSNVGE SEQ
ID NO: 130
CDR3a CAATSRDDMRF SEQ
ID NO: 131
Variable mTsiRAvFIFLwLQLDINNGENvEQHpsTLsvQEGDsAviKc SEQ ID NO:
132
TY SD SASNYFPWYKQELGKRPQLI ID IRSNVGEKKDQRIAVT
LNKTAKHFSLHI TETQPEDSAVYFCAATSRDDMRFGAGTRLT
VKPN
Full ¨ with MT S I RAVF IFLWLQLDLVNGENVEQHP S TLSVQEGD SAVI KC SEQ ID NO: 133
TRAC constant TY SD SASNYFPWYKQELGKRPQLI ID IRSNVGEKKDQRIAVT
domain LNKTAKHFSLHI TETQPEDSAVYFCAATSRDDMRFGAGTRLT
VKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKD
SDVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS I
IP EDTFFP SP ES SCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
Beta (13) 1 CDR1I3 SEHNR
SEQ ID NO: 134
CDR2I3 FQNEAQ SEQ
ID NO: 135
CDR3I3 CASSLEGRAMPRDSHQETQYF SEQ
ID NO: 136
Variable mGTsLLcwmALcLLGADHADTGvsQNpRHKITKRGQNvTFRc SEQ ID NO:
137
DP I SEHNRLYWYRQTLGQGP EFLTYFQNEAQLEKSRLLSDRF
SAERPKGSFSTLEI QRTEQGDSAMYLCASSLEGRAMPRDSHQ
ETQYFGPGTRLLVLE
Full ¨ with MGTSLLCWMALCLLGADHADTGVS QNPRHK I TKRGQNVTFRC SEQ ID NO: 138
TRBC1 DP I SEHNRLYWYRQTLGQGP EFLTYFQNEAQLEKSRLLSDRF
constant SAERPKGSFSTLEI QRTEQGDSAMYLCASSLEGRAMPRDSHQ
domain ETQYFGPGTRLLVLEDLNKVFPPEVAVFEP SEAE I SHTQKAT
LVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN
DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQD
RAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSAT ILYEILLG
KATLYAVLVSALVLMAMVKRKDF
Full ¨ with MGTSLLCWMALCLLGADHADTGVS QNPRHK I TKRGQNVTFRC SEQ ID NO: 139
TRBC2 DP I SEHNRLYWYRQTLGQGP EFLTYFQNEAQLEKSRLLSDRF
constant SAERPKGSFSTLEI QRTEQGDSAMYLCASSLEGRAMPRDSHQ
domain ETQYFGPGTRLLVLEDLKNVFPPEVAVFEP SEAE I SHTQKAT
LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN
DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQD
RAKPVTQIVSAEAWGRADCGFT SE SYQQGVLSAT ILYEILLG
KATLYAVLVSALVLMAMVKRKDSRG
Beta (13) 2 CDR1I3 LNHNV SEQ
ID NO: 140
CDR2I3 YYDKDF SEQ
ID NO: 141
CDR3I3 CATSWGLNEQYF SEQ
ID NO: 142
Variable mGPGLLHwmALcLLGTGHGDAmviQNpRyQvTQFGKpvTLsc SEQ ID NO:
143
SQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQ
SRRPNTSFCFLDIRSPGLGDAAMYLCATSWGLNEQYFGPGTR
LTVTE
Full ¨ with MGPGLLHWMALCLLGTGHGDAMVI QNPRYQVTQFGKPVTLSC SEQ ID NO: 144
TRBC1 SQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQ
constant SRRPNTSFCFLDIRSPGLGDAAMYLCATSWGLNEQYFGPGTR
domain LTVTEDLNKVFPPEVAVFEP SEAE I S HTQKATLVCLATGFFP
DHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSVSYQQGVLSAT ILYEILLGKATLYAVLVS
ALVLMAMVKRKDF
Full ¨ with MGPGLLHWMALCLLGTGHGDAMVI QNPRYQVTQFGKPVTLSC SEQ ID NO: 145
TRBC2 SQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQ
constant SRRPNTSFCFLDIRSPGLGDAAMYLCATSWGLNEQYFGPGTR
domain LTVTEDLKNVFPPEVAVFEP SEAE I S HTQKATLVCLATGFYP
DHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL
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RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSESYQQGVLSAT ILYEILLGKATLYAVLVS
ALVLMAMVKRKDSRG
Patient 3
Chain Region Amino acid sequence SEQ ID NO
Alpha (a) CDR1a TRDTTYY SEQ ID NO: 146
1 CDR2a RNSFDEQN SEQ ID NO: 147
CDR3a CALPDKVIF SEQ ID NO: 148
Variable mLTAsLLRAviAsicvvssmAuvTQAQTEisvvEKEDvTLD SEQ ID NO: 149
CVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEI SGRY
SWNFQKSTSSFNFT I TAS QVVDSAVYFCALPDKVIFGP GT SL
SVIPN
Full ¨ with MLTASLLRAVIAS I CVVS SMAQKVTQAQTE I SVVEKEDVTLD SEQ ID NO: 150
TRAC constant CVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEI SGRY
domain SWNFQKSTSSFNFT I TAS QVVDSAVYFCALPDKVIFGP GT SL
SVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK
DS DVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS
I I PEDTFFP SPESSCDVKLVEKSFETDTNLNFQNLSVI GFRI
LLLKVAGFNLLMTLRLWSS
Alpha (a) CDR1a S I FNT SEQ ID NO: 151
2 CDR2a LYKAGEL SEQ ID NO: 152
CDR3a CAGLYATNKL I F SEQ ID NO: 153
Variable MLLEHLLI ILWMQLTWVSGQQLNQSP QSMF IQEGEDVSMNCT SEQ ID NO:
154
SS S I FNTWLWYKQEPGEGPVLL IALYKAGELT SNGRLTAQFG
I TRKDSFLNI SAS I P SDVGI YFCAGLYATNKLIFGTGTLLAV
QPN
Full ¨ with =EH= iLwmQLTWVSGQQLNQSPQSMF IQEGEDVSMNCT SEQ ID NO: 155
TRAC constant sss I FNTWLWYKQEPGEGPVLL IALYKAGELT SNGRLTAQFG
domain I TRKDSFLNI SAS I P SDVGI YFCAGLYATNKLIFGTGTLLAV
QPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS
DVY I TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS II
PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILL
LKVAGFNLLMTLRLWSS
Beta (13) CDR113 SGDLS SEQ ID NO: 156
CDR213 YYNGEE SEQ ID NO: 157
CDR313 CASSVSAGSTGELFF SEQ ID NO: 158
Variable mGFRLLccvAFcLLGAGPvDsGyrupKHLITATGQRvTLRc SEQ ID NO: 159
SP RSGDLSVYWYQQSLDQGLQFLI QYYNGEERAKGNILERFS
AQQFPDLHSELNLSSLELGDSALYFCASSVSAGSTGELFFGE
GS RLTVLE
Full ¨ with MGFRLLCCVAFCLLGAGPVDSGVTQTpKHL I TATGQRVTLRC SEQ ID NO: 160
TRBC1 SP RSGDLSVYWYQQSLDQGLQFLI QYYNGEERAKGNILERFS
constant AQQFPDLHSELNLSSLELGDSALYFCASSVSAGSTGELFFGE
domain GSRLTVLEDLNKVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTSVS YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDF
Full ¨ with MGFRLLCCVAFCLLGAGPVDSGVTQTpKHL I TATGQRVTLRC SEQ ID NO: 161
TRBC2 SP RSGDLSVYWYQQSLDQGLQFLI QYYNGEERAKGNILERFS
constant AQQFPDLHSELNLSSLELGDSALYFCASSVSAGSTGELFFGE
domain GSRLTVLEDLKNVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTS ES YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDS RG
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Accordingly, the present invention provides isolated polypeptides comprising
one or more
amino acid sequences selected from the group consisting of SEQ ID NOs: 1-55,
91-161,
182-191, 194-203 and 214-217, fragments, variants and homologues thereof.
In one aspect, the invention provides a TCR comprising a TCR alpha chain
sequence
-- selected from the group consisting of the HD12-HD15 alpha chain sequences
of Table 1,
and a TCR beta chain sequence independently selected from the group consisting
of the
HD12-H D15 beta chain sequences of Table 1.
In one aspect, the invention provides a TCR comprising a TCR alpha chain
sequence
selected from the group consisting of the Patient 1, Patient 2 or Patient 3
alpha chain
-- sequences of Table 1, and a TCR beta chain sequence independently selected
from the
group consisting of the Patient 1, Patient 2 or Patient 3 beta chain sequences
of Table 1.
In one aspect, the invention provides a TCR comprising a TCR alpha chain
sequence
selected from the group consisting of the HD12, HD13, HD14, HD15, Patient 1,
Patient 2 or
Patient 3 alpha chain sequences of Table 1, and a TCR beta chain sequence
independently
-- selected from the group consisting of the HD12, HD13, HD14, HD15, Patient
1, Patient 2 or
Patient 3 beta chain sequences of Table 1.
In alternative embodiments, sequences of the full TCR beta chains referred to
in Table 1
may be replaced with corresponding sequences below.
Donor: HD12
Beta() Full ¨ with mDTwiNcwAiFsLLKAGLTEpEvTQTpsHQvTQmGQEviLRc SEQ ID
NO: 222
TRBC1 VP I SNHLYFYWYRQ ILGQKVEFLVSFYNNE I SEKSE IFDDQF
constant SVERPDGSNFTLKI RS TKLEDSAMYFCASSEAGLSYEQYFGP
domain GTRLTVTEDLNKVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTSVS YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDF
-- In some embodiments, SEQ ID NO: 10 may be replaced with SEQ ID NO: 222.
Donor: HD13
Beta() Full ¨ with MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLEC SEQ ID
NO: 223
TRBC1 SQTKGHDRMYWYRQDP GLGLRL I YYSFDVKDINKGE I SDGYS
constant VSRQAQAKFSLSLE SAIPNQTALYFCATGQATQETQYFGP GT
domain RLLVLEDLNKVFPPEVAVFEPSEAEI SHTQKATLVCLATGFF
PDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SR
LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ IV
SAEAWGRADCGFTSVS YQQGVLSAT I LYE I LLGKATLYAVLV
SALVLMAMVKRKDF
In some embodiments, SEQ ID NO: 21 may be replaced with SEQ ID NO: 223.
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Donor: HD14
Beta() Full ¨ with MGFRLLCCVAFCLLGAGPVD SGVTQTPKHL I TATGQRVTLRC SEQ ID
NO: 224
TRBC1 SP RS GDLSVYWYQQSLDQGLQFLI QYYNGEERAKGNILERFS
constant AQQFPDLHSELNLS SLELGDSALYFCASGRGDTEAFFGQGTR
domain LTVVEDLNKVFPPEVAVFEP SEAE I S HTQKATLVCLATGFFP
DHVELSWWVNGKEVHS GVSTDP QP LKEQPALNDSRYCLS SRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSVSYQQGVLSAT ILYE ILLGKATLYAVLVS
ALVLMAMVKRKDF
In some embodiments, SEQ ID NO: 32 may be replaced with SEQ ID NO: 224.
Donor: HD15
Beta() Full ¨ with MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKS SEQ ID NO:
225
S4 TRBC1 LKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPS
populatio constant RF SP ECPNS S HLFLHLHTLQPEDSALYLCAS S QEEGAVYGYT
n domain FGSGTRLTVVEDLNKVFPPEVAVFEP SEAE I S
HTQKATLVCL
ATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY
CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADCGFTSVSYQQGVLSAT ILYE ILLGKATL
YAVLVSALVLMAMVKRKDF
Beta() Full ¨ with MGPGLLHWMALCLLGTGHGDAMVI QNPRYQVTQFGKPVTLSC SEQ ID
NO: 226
S1 IFNg TRBC1 SQTLNHNVMYWYQQKS SQAPKLLFHYYDKDFNNEADTPDNFQ
enriched constant SRRPNTSFCFLD IRSPGLGDAAMYLCATSREGLAADTQYFGP
populatio domain GTRLTVLEDLNKVFPP EVAVFEP S EAE I SHTQKATLVCLATG
n FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGF TSVS YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDF
In some embodiments, SEQ ID NO: 43 may be replaced with SEQ ID NO: 225.
In some embodiments, SEQ ID NO: 54 may be replaced with SEQ ID NO: 226.
Patient 1
Beta (13) 1 Full ¨ with MGTSLLCWMALCLLGADHADTGVS QNPRHK I TKRGQNVTFRC SEQ ID
NO: 227
direct TRBC1 DP I S EHNRLYWYRQTLGQGP EFLTYFQNEAQLEKSRLLSDRF
sequenci constant SAERPKGSFS TLE I QRTEQGDSAMYLCASS SGLAFYEQYFGP
ng upon domain GTRLTVTEDLNKVFPP EVAVFEP S EAE I SHTQKATLVCLATG
sorting FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGF TSVS YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDF
Beta (13) 2 Full ¨ with MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRC SEQ ID NO:
228
direct TRBC1 DP I S GHDNLYWYRRVMGKE I KFLLHFVKESKQDE SGMPNNRF
sequenci constant LAERTGGTYSTLKVQPAELEDSGVYFCASSQLSGRDSYEQYF
ng upon domain GP GTRLTVTEDLNKVFPP EVAVFEP S EAE I SHTQKATLVCLA
sorting TGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYC
LS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV
TQ IVSAEAWGRADCGF TSVS YQQGVLSAT I LYE I LLGKATLY
AVLVSALVLMAMVKRKDF
Alpha (a) Full ¨ with MGPQLLGYVVLCLLGAGP LEAQVTQNPRYL I TVTGKKLTVTC SEQ ID NO:
229
2 TRBC1 SQNMNHEYMSWYRQDP GLGLRQ I YYSMNVEVTDKGDVP EGYK
growing constant VSRKEKRNFP LI LE SP SPNQTSLYFCAS STLGGELFFGEGSR
colony domain LTVLEDLNKVFPPEVAVFEP SEAE I S HTQKATLVCLATGFFP
DHVELSWWVNGKEVHS GVSTDP QP LKEQPALNDSRYCLS SRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSVSYQQGVLSAT ILYE ILLGKATLYAVLVS
ALVLMAMVKRKDF
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In some embodiments, SEQ ID NO: 100 may be replaced with SEQ ID NO: 227.
In some embodiments, SEQ ID NO: 106 may be replaced with SEQ ID NO: 228.
In some embodiments, SEQ ID NO: 122 may be replaced with SEQ ID NO: 229.
Patient 2
Beta (13) 1 Full ¨ with MGTSLLCWMALCLLGADHADTGVS QNPRHK I TKRGQNVTFRC SEQ ID
NO: 230
TRBC1 DP I SEHNRLYWYRQTLGQGP EFLTYFQNEAQLEKSRLLSDRF
constant SAERPKGSFSTLEI QRTEQGDSAMYLCAS SLEGRAMPRDS HQ
domain ETQYFGPGTRLLVLEDLNKVFPPEVAVFEP SEAE I S HTQKAT
LVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN
DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQD
RAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSAT ILYE ILLG
KATLYAVLVSALVLMAMVKRKDF
Beta (13) 2 Full ¨ with MGPGLLHWMALCLLGTGHGDAMVI QNPRYQVTQFGKPVTLSC SEQ ID NO:
231
TRBC1 SQTLNHNVMYWYQQKS SQAPKLLFHYYDKDFNNEADTPDNFQ
constant SRRPNTSFCFLD IRSPGLGDAAMYLCATSWGLNEQYFGPGTR
domain LTVTEDLNKVFPPEVAVFEP SEAE I S HTQKATLVCLATGFFP
DHVELSWWVNGKEVHS GVSTDP QP LKEQPALNDSRYCLS SRL
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSVSYQQGVLSAT ILYE ILLGKATLYAVLVS
ALVLMAMVKRKDF
In some embodiments, SEQ ID NO: 138 may be replaced with SEQ ID NO: 230.
In some embodiments, SEQ ID NO: 144 may be replaced with SEQ ID NO: 231.
Patient 3
Beta() Full ¨ with MGFRLLCCVAFCLLGAGPVD SGVTQTPKHL I TATGQRVTLRC SEQ ID
NO: 232
TRBC1 SP RS GDLSVYWYQQSLDQGLQFLI QYYNGEERAKGNILERFS
constant AQQFPDLHSELNLS SLELGDSALYFCAS SVSAGSTGELFFGE
domain GSRLTVLEDLNKVFPPEVAVFEPSEAEI SHTQKATLVCLATG
FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS
SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGF TSVS YQQGVLSAT I LYE I LLGKATLYAV
LVSALVLMAMVKRKDF
In some embodiments, SEQ ID NO: 160 may be replaced with SEQ ID NO: 232.
Reduced mispairing and improved TCR expression
The TCR of the invention may be expressed in a T-cell to alter the antigen
specificity of the
T-cell. TCR-transduced T-cells may express at least two TCR alpha and two TCR
beta
chains. While the endogenous TCR alpha/beta chains form a receptor that is
self-tolerant,
the introduced TCR alpha/beta chains form a receptor with defined specificity
for the given
target antigen.
However, TCR gene therapy requires sufficient expression of transferred TCRs.
Trasferred
TCR might be diluted by the presence of the endogeneous TCR, resulting in
suboptimal
expression of the tumor specific TCR. Furthermore, mispairing between
endogenous and
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introduced chains may occur to form novel receptors, which might display
unexpected
specificities for self-antigens and cause autoimmune damage when transferred
into patients.
Hence, several strategies have been explored to reduce the risk of mispairing
between
endogenous and introduced TCR chains. Mutations of the TCR alpha/beta
interface is one
strategy currently employed to reduce unwanted mispairing. For example, the
introduction
of a cysteine in the constant domains of the alpha and beta chain allows the
formation of a
disulfide bond and enhances the pairing of the introduced chains while
reducing mispairing
with wild type chains.
Accordingly, the TCRs of the invention may comprise one or more mutations at
the a chain/6
chain interface, such that when the a chain and the 13 chain are expressed in
a T-cell, the
frequency of mispairing between said chains and endogenous TCR a and 13 chains
is
reduced. In one embodiment, the one or more mutations introduce a cysteine
residue into
the constant region domain of each of the a chain and the 13 chain, wherein
the cysteine
residues are capable of forming a disulphide bond between the a chain and the
13 chain.
Such modification of TCRs is described in for example Boulter, J.M et al.
(2003) Protein
Engineering 16: 707-711 and Kuball, L. et al. (2007) Blood 109: 2331-8.
In one embodiment, the one or more mutations are at amino acid positions
selected from
those disclosed in Table 1 of Boulter, J.M et al. (2003) Protein Engineering
16: 707-711. In
one embodiment, the one or more mutations are a substitution of one or more of
the
following amino acids with cysteine:
TR residue 1[[1,[ sidue
Thr...:41ine 48 1)
conine 45
Seville 61 .-,erine 57
-[icine 50 [,erine 57
,osine 1( '[-erine
8, rine 15 aline 13
[ ine 15 Glutamate 15
[[reonine 45 Asparlate
'eine 12 Serine 17
[ inc I Aq_,,inine 79
.2 IThenylalimint I
[inc 22 Phenylalanin, 1 [
Tyrosine 43 1.encine 63
In a one embodiment, the TCR comprises one or more of the following groups of
mutations:
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(a) a substitution of threonine at position 48 of the TCR alpha constant gene
with
cysteine; and/or a substitution of serine at position 57 of the TCR beta
constant gene
with cysteine;
(b) a substitution of threonine at position 45 of the TCR alpha constant gene
with
cysteine; and/or a substitution of serine at position 77 of the TCR beta
constant gene
with cysteine;
(c) a substitution of serine at position 61 of the TCR alpha constant gene
with cysteine;
and/or a substitution of serine at position 57 of the TCR beta constant gene
with
cysteine;
(d) a substitution of leucine at position 50 of the TCR alpha constant gene
with cysteine;
and/or a substitution of serine at position 57 of the TCR beta constant gene
with
cysteine;
(e) a substitution of tyrosine at position 10 of the TCR alpha constant gene
with cysteine;
and/or a substitution of serine at position 17 of the TCR beta constant gene
with
cysteine;
(f) a substitution of serine at position 15 of the TCR alpha constant gene
with cysteine;
and/or a substitution of valine at position 13 of the TCR beta constant gene
with
cysteine;
(g) a substitution of serine at position 15 of the TCR alpha constant gene
with cysteine;
and/or a substitution of glutamate at position 15 of the TCR beta constant
gene with
cysteine;
(h) a substitution of threonine at position 45 of the TCR alpha constant gene
with
cysteine; and/or a substitution of aspartate at position 59 of the TCR beta
constant
gene with cysteine;
(i) a substitution of leucine 12 at position 48 of the TCR alpha constant gene
with
cysteine; and/or a substitution of serine at position 17 of the TCR beta
constant gene
with cysteine;
(j) a substitution of serine at position 61 of the TCR alpha constant gene
with cysteine;
and/or a substitution of arginine at position 79 of the TCR beta constant gene
with
cysteine;
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(k) a substitution of leucine at position 12 of the TCR alpha constant gene
with cysteine;
and/or a substitution of phenylalanine at position 14 of the TCR beta constant
gene
with cysteine;
(I) a substitution of valine at position 22 of the TCR alpha constant gene
with cysteine;
and/or a substitution of phenylalanine at position 14 of the TCR beta constant
gene
with cysteine; and/or
(m)a substitution of tyrosine at position 43 of the TCR alpha constant gene
with cysteine;
and/or a substitution of leucine at position 63 of the TCR beta constant gene
with
cysteine.
In a preferred embodiment, the TCR comprises a substitution of threonine at
position 48 of
the TCR alpha constant gene with cysteine; and/or a substitution of serine at
position 57 of
the TCR beta constant gene with cysteine.
Another strategy to reduce mispairing relies on the introduction of
polynucleotide sequences
encoding siRNA, added to the genes encoding for the tumor specific TCR a and
or 13 chains,
and designed to limit the expression of the endogenous TCR genes (Okamoto S.
Cancer
research 69, 9003-9011, 2009).
Accordingly, the vector or polynucleotide encoding the TCRs of the invention
may comprise
one or more siRNA or other agents aimed at limiting or abrogating the
expression of the
endogenous TCR genes.
It is also possible to combine artificial nucleases, such as zinc finger
nucleases (ZFN),
transcription activator-like effector nucleases (TALEN) or CRISPR/Cas systems,
designed to
target the constant regions of the endogenous genes, e.g. TCR genes (TRAC and,
or
TRBC), to obtain the permanent disruption of the endogenous TCR alpha and/or
beta chain
genes, thus allowing full expression of the tumor specific TCR and thus
reducing or
abrogating the risk of TCR mispairing. This process, known as the TCR gene
editing proved
superior to TCR gene transfer in vitro and in vivo (Provasi E., Genovese P.,
Nature Medicine
May; 18(5):807-15; 2012; Mastaglio S. et al. (2017) Blood 130: 606-618).
Accordingly, the TCRs of the invention may be used to edit T cell specificity
by TCR
disruption and genetic addition of the tumor specific TCR.
In addition, the genome editing technology allows targeted integration of a
expression
cassette, comprising a polynucleotide encoding a TCR of the invention, and
optionally one or
more promoter regions and/or other expression control sequences, into an
endogenous
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gene disrupted by the artificial nucleases (Lombardo A., Nature biotechnology
25, 1298-
1306; 2007).
Accordingly, the TCRs of the invention may be used to edit T-cell specificity
by targeted
integration of a polynucleotide encoding a TCR of the invention at a genomic
region. The
integration may be targeted by an artificial nuclease.
A cell, such as a T cell, may therefore be genetically engineered to comprise
a TCR of the
invention. In addition, a cell, such as a T cell, may be genetically edited by
gene disruption,
for example TRAC and/or TRBC disruption obtained by, for example, CRISPR/Cas9,
or by
targeted integration, for example of an expression cassette into an endogenous
gene (such
as an endogenous gene involved in antigen specificity, persistence, expansion,
activity,
resistance to exhaustion/senescence/inhibitory signals, homing capacity or
other T-cell
functions).
Another strategy developed to increase expression of the transferred TCR and
to reduce
TCR mispairing consists in "murinization," which replaces the human TCR a and
TCR 13
constant regions (e.g. the TRAC, TRBC1 and TRBC2 regions) by their murine
counterparts.
Murizination of TCR constant regions is described in, for example, Sommermeyer
and
Uckert J Immunol; 2010 (184:6223-6231). Accordingly, the TCRs of the invention
may be
murinized.
Isolated polynucleotide
The invention relates to an isolated polynucleotide encoding a TCR of the
invention or a part
thereof, such as the a chain and/or the 13 chain, a variable domain or a
portion thereof.
The isolated polynucleotide may be double or single stranded, and may be RNA
or DNA.
It will be understood by a skilled person that numerous different
polynucleotides can encode
the same polypeptide as a result of the degeneracy of the genetic code. In
addition, it is to
be understood that the skilled person may, using routine techniques, make
nucleotide
substitutions, additions or deletions that do not affect the polypeptide
sequence encoded by
the polynucleotides of the invention to reflect the codon usage of any
particular host
organism in which the polypeptides of the invention are to be expressed.
The polynucleotides described herein may be modified by any method available
in the art.
.. Such modifications may be carried out in order to enhance the in vivo
activity or lifespan of
the polynucleotides of the invention.
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Polynucleotides such as DNA polynucleotides may be produced recombinantly,
synthetically
or by any means available to those of skill in the art. They may also be
cloned by standard
techniques.
Longer polynucleotides will generally be produced using recombinant means, for
example
using polymerase chain reaction (PCR) cloning techniques. This will involve
making a pair
of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence
which it is
desired to clone, bringing the primers into contact with mRNA or cDNA obtained
from an
animal or human cell, performing a polymerase chain reaction under conditions
which bring
about amplification of the desired region, isolating the amplified fragment
(e.g. by purifying
the reaction mixture with an agarose gel) and recovering the amplified DNA.
The primers
may be designed to contain suitable restriction enzyme recognition sites so
that the
amplified DNA can be cloned into a suitable vector.
Examples of nucleotide sequences encoding TCRs according to the invention are
provided
in the Table 2.
Table 2
Donor Chain Nucleotide sequence SEQ ID NO
HD12 a (with A1C,1(_,ACII11(liAL,C_CilL,CilL,I\AL,-1(_,AAL,-
(il1(_,ACil GtGG LAC,C,AC_CilL, SEQ ID NO: 56
TRAC) GCATIGCCCAGAAGATAACICAAACCCAACCAGGAAIGIT CGIGCAGGAAAAGGA
GGC T GT GAC T C T GGAC T GCACATAT GACACCAGT GAT CAAAGT TAT GGT C TAT TC
TGGTACAAGCAGCCCAGCAGTGGGGAAATGATTITTOTTATITATCAGGGGTCTI
AT GACGAGCAAAAT GCAACAGAAGGT CGC TAC T CAT T GAAT T T CCAGAAGGCAAG
AAAAT CCGCCAACC T T GT CAT C T CCGC T T CACAAC T GGGGGAC T CAGCAAT GTAT
T TCTGTGCAATGAGAACGGGAGGAGGIGCTGACGGACTCACCTTTGGCAAAGGGA
CTCATCTAATCATCCAGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCT
GAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCT
CAAACAAAT GT GT CACAAAGTAAGGAT T C T GAT GT GTATAT CACAGACAAAAC T G
T GC TAGACAT GAGGT C TAT GGAC T T CAAGAGCAACAGT GC T GT GGCC T GGAGCAA
CAAATCTGACT T T GCAT GT GCAAACGCC T TCAACAACAGCAT TAT TCCAGAAGAC
ACCT TCT TCCCCAGCCCAGAAAGT TCCTGTGATGTCAAGCTGGTCGAGAAAAGC T
T TGAAACAGATACGAACCTAAACTT T CAAAACC T GT CAGT GAT T GGGT T CCGAAT
CCTCCTCCTGAAACTMCCGCCTITAATCTGCTCATGACGCTGCCGCTGTCGTCC
AGC
a (with ATGAGCC TGTC TAGC CT GC TGAAGGTGGT CACAGC CAGC CT GT
GGCT CG SEQ ID NO: 204
TRAC) GAC CT GGAATC GC CCAGAAGATCAC CCAGACACAGCC CGGCAT GT TC
GT
GCAAGAGAAAGAAGCCGTGACACTGGACTGCACCTACGACACCAGCGAT
CAGAGCTAC GGCC TGTT CT GGTACAAGCAGC CTAGCAGC GGCGAGAT GA
T CT TC CT GATC TACCAGGGCAGC TACGAC GAGCAGAATGCCAC CGAGGG
CAGATACAGCC TGAACT TC CAGAAGGC CC GGAAGT CC GC CAAC CT GGTC
ATT TC TGCT TC TCAGCT GGGC GACAGC GC CATGTACT TT TGCGCCAT GA
GAACAGGCGGC GGAGCC GATGGACT GACATT TGGCAAGGGCAC CCAC CT
GAT CATC CAGC CT TACATT CAGAAC CC CGAT CC TGCC GT GTAC CAGC TG
AGGGATAGCAAGAGCAGCGACAAGAGC GT GT GC CT GT TCAC CGAC TT CG
ACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCAC
C GATAAGTGCGTGCT GGACAT GC GGAGCATGGACT TCAAGAGCAACAGC
GCC GT GGCC TGGT CCAACAAGAGCGAT TT CGCC TGCGCCAACGCC TT CA
ACAACAGCATTAT CC CC GAGGACACAT TC TT CC CAAGTC CT GAGAGCAG
C TGCGAC GT GAAGCT GGTGGAAAAGAGCT TC GAGACAGACACCAACC TC
AAT TT CCAGAACC TGAGCGTGAT CGGC TT CC GGAT CC TGCT GC TGAAAG
T GGCC GGCT TCAACC TGCT GATGAC CC TGAGAC TGTGGT CCAGC
13 (with ATGGATACCTGGCTCGTATGCTGGGCAAT T T T TAGTCTCT
TGAAAGCAGGACTCA SEQ ID NO: 57
TRBC1) CAGAACCIGAAGICACCCAGACTCCCAGCCATCAGGICACACAGAIGGGACAGGA
AGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATTGGTACAGA
CAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAATAATGAAATCT
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CAGAGAAGTCTGAAATAT TCGATGATCAAT TCTCAGT TGAAAGGCCTGATGGATC
AAATIT CAC C I GAAGAT CCGG I CCACAAAGC T GGAGGAC T CAGCCAT G TACT TC
T GT GCCAGCAGT GAAGCGGGAC T T T CC TACGAGCAGTAC T TCGGGCCGGGCACCA
GGCTCACGGTCACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTT
T GAGCCAT CAGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC TG
GCCACAGGCT TCT TCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG
AGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAAT GAC T CCAGATAC T GCC T GAGCAGCCGCC T GAGGGT CT CGGCCACC T TCTGG
CAGAACCCCCGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGCTCTCGGAGA
AT GACGAGT GGACCCAGGATAGGGCCAAACCCGT CACCCAGAT CGT CAGCGCCGA
GGCC T GGGGTAGAGCAGAC T GT GGC T T TACC T CGGT GT CC TACCAGCAAGGGGT C
C T GTCTGCCACCATCCTC T AT GAGATCCTGCTAGGGAAGGCCACCCTGT AT GCT G
TGCTGGTCAGCGCCCTIGIGTTGATGGCCATGGICAAGAGAAAGGATTIC
13 (with ATGGATACCTGGCTCGTATGCTGGGCAATTITTAGICTCTTGAAAGCAGGACTCA SEQ
ID NO: 58
TRBC2) CAGAAC CTGAAGT CAC CCAGAC T CCCAGC CAT CAGGICACACAGAT
GGGACAGGA
AGTGATCT TGCGCTGTGTCCCCATCTCTAATCACT TATACT TCTAT TGGTACAGA
CAAATCT TGGGGCAGAAAGTCGAGT T TCTGGT T TCCT T T TATAATAATGAAATCT
CAGAGAAGTCTGAAATAT T CGAT GAT CAAT TCTCAGT T GAAAGGCC T GAT GGAT C
AAAT T T CAC T C T GAAGAT CCGGT CCACAAAGC T GGAGGAC T CAGCCAT GTAC T TC
T GT GCCAGCAGT GAAGCGGGAC T T T CC TACGAGCAGTAC T TCGGGCCGGGCACCA
GGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTT
T GAGCCAT CAGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC TG
GCCACAGGCT TCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG
AGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCT TCTGG
CAGAACCCCCGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGCTCTCGGAGA
AT GACGAGT GGACCCAGGATAGGGCCAAACC T GT CACCCAGAT CGT CAGCGCCGA
GGCC T GGGGTAGAGCAGAC T GT GGC T TCACCTCCGAGTCT TACCAGCAAGGGGTC
CTGTCTGCCACCATCCTCTATGAGATCT TGCTAGGGAAGGCCACCT TGTATGCCG
T GC T GGT CAGT GCCC T CGT GC T GAT GGCCAT GGT CAAGAGAAAGGAT TCCAGAGG
13 (with ATGGATACTTGGCTTGTGTGCTGGGCCATCTTCAGCCTGCTGAAGGCCG SEQ ID NO:
205
TRBC2) GAC TGACAGAGCC CGAAGT GACACAGACACC CAGC CACCAAGT GACC CA
GAT GGGC CAAGAAGT GATC CT GC GC TGCGTGCC CATCAGCAAC CACC TG
TAO TT CTAC TGGTACAGACAGAT CC TGGGCCAGAAAGTGGAAT TO CT GG
T GT CC TT CTACAACAAC GAGATCAGCGAGAAGT CC GAGATC TT CGAC GA
CCAGT TCAGCGTGGAAAGACCCGACGGCAGCAACT TCAC CC TGAAGATC
AGAAGCACCAAGC TO GAGGACAGCGCCAT GTAC TT TT GC GC CT CT TO TG
AAGCCGGCCTGAGCTACGAGCAGTACT T T GGCC CT GGCACCAGAC TGAC
C GT GACC GAGGAT CT GAAGAACGTGT T CC CACC TGAGGT GGCC GT GT TO
GAACC TT CT GAGGCC GAGATC TO TCACAC CCAGAAAGCCACAC TO GT GT
GTCTGGCCACCGGCT TO TATC CC GATCAC GT GGAACT GT CT TGGT GGGT
CAACGGCAAAGAGGT GCACAGCGGC GT CT GTAC CGAT CC TCAGCC TO TG
AAAGAGCAGCC CGCT CT GAAC GACAGCAGATAC TGCC TGAGCAGCAGAC
T GAGAGT GT CC GC CACC TT CT GGCAGAAC CC CAGAAACCAC TT CAGATG
CCAGGTGCAGT TO TACGGC CT GT CO GAGAAC GATGAGTGGACC CAGGAT
AGAGCCAAGCCTGTGACTCAGATCGTGTCTGCCGAAGCCTGGGGCAGAG
CCGAT TGTGGCTT TACCAGCGAGAGCTAC CAGCAGGGCGTGCT GT CT GC
CACAATC CT GTAC GAGATC CT GC TGGGCAAAGC CACT CT GTAC GC CGTG
C TGGT GT CT GC CC TGGT GC TGAT GGCCAT GGTCAAGC GGAAGGATAGCA
GGGGC
Donor Chain Nucleotide sequence SEQ ID NO
HD13 a (with A1GAAG11GG1GACAAGCA11AC1G1AC1CC1A1C111GGG1A11A1GGG1GA1C,
SEQ ID NO: 59
TRAC) CTAAGACCACACAGCCAAATICAAIGGAGAGIAACGAAGAAGAGCCIGTICACIT
GCCT T GTAACCAC T CCACAAT CAGT GGAAC T GAT TACATACAT TGGTATCGACAG
CT T CCC T CCCAGGGT CCAGAGTACGT GAT T CAT GGT C T TACAAGCAAT GT GAACA
ACAGAATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTCCAGTACCT TGAT CC T
GCACCGTGCTACCTTGAGAGATGCTGCTGTGTACTACTGCATCCTGAGTACCCGG
GTCTGGGCTGGGAGT TACCAACTCACT T TCGGGAAGGGGACCAAACTCTCGGT CA
TACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATC
CAGTGACAAGTCTGTCTGCCTAT TCACCGAT T T TGAT TCTCAAACAAATGTGTCA
CAAAGTAAGGAT T C T GAT GT GTATAT CACAGACAAAAC T GT GC TAGACAT GAGG T
C TAT GGAC T T CAAGAGCAACAGT GC T GT GGCC T GGAGCAACAAAT C T GAC T T T GC
AT GT GCAAACGCCT TCAACAACAGCAT TAT TCCAGAAGACACCT TCT TCCCCAGC
CCAGAAAGT T CC T GT GAT GT CAAGC T GGT CGAGAAAAGC T T TGAAACAGATACGA
ACCTAAACTT TCAAAACCTCTCAGTCATTCCCTTCCGAATCCTCCTCCTCAAAGT
GGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
a (with ATGAAGCTGGTCACCAGCATCACCGTGCTGCTGAGCCTGGGCATTATGG SEQ ID NO:
206
TRAC) G C GAC GC CAAGAC CACACAGC C CAACAGCAT GGAAAG CAAC GAAGAG GA
ACC CGTGCATC TGCC CT GCAACCACAGCACAAT CAGC GGCACC GACTAC
ATC CACT GGTACAGACAGC TGCC CAGC CAGGGACC TGAGTATGTGAT CC
ACGGC CT GACCAGCAAC GT GAACAACAGAAT GGCCAGCC TGGC TATC GC
C GAGGACAGAAAGAGCAGCAC CC TGAT CC TGCACAGAGC CACACT GAGA
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GATGCCGCCGTGTACTACTGCATCCTGAGCACAAGAGTGTGGGCCGGCA
GCTACCAGCTGACATTTGGCAAGGGCACCAAGCTGAGCGTGATCCCCAA
CATTCAGAACCCCGATCCTGCCGTGTACCAGCTGCGGGATAGCAAGAGC
AGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACG
TGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCT
GGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCC
AACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCC
CTGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAACT
GGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTG
TCCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGGCTTCAACC
TGCTGATGACCCTGAGACTGTGGTCCAGC
13 (with ATGGCCTCCCTGCTCTTCTTCTGTGGGGCCTTTTATCTCCTGGGAACAGGGTCCA SEQ
ID NO: 60
TRBC1) TGGAIGCT GAT G I TAC C CAGACCCCAAGGAATAGGAT
CACAAAGACAGGAAAGAG
GAT TAT GC T GGAAT GT T C T CAGAC TAAGGGT CAT GATAGAAT GTAC T GGTAT CGA
CAAGACCCAGGACTGGGCCTACGGT T GAT C TAT TAC T CC T T T GAT GT CAAAGATA
TAAACAAAGGAGAGATCTCTGAT GGATACAGT GTCTCTCGACAGGCACAGGC TAA
AT TCTCCCTGTCCCTAGAGTCTGCCATCCCCAACCAGACAGCTCT T TACT TCTGT
GCCACCGGCCAGGCGACCCAAGAGACCCAGTACT TCGGGCCAGGCACGCGGCTCC
TGGTGCTCGAGGACCTGAACAAGGTGT TCCCACCCGAGGTCGCTGTGT T TGAGCC
AT CAGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC T GGCCACA
GGCT TCT TCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGC
ACAGT GGGGT CAGCACGGACCCGCAGCCCC T CAAGGAGCAGCCCGCCC T CAAT GA
CTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCT TCTGGCAGAAC
CCCCGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGCTCTCGGAGAATGACG
AGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTG
GGGTAGAGCAGACTGTGGCT T TACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCT
GCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGG
TCAGCGCCCT T GT GT T GAT GGCCAT GGT CAAGAGAAAGGAT T T C
13 (with ATGGCCTCCCTGCTCTTCTTCTGTGGGGCCTTTTATCTCCTGGGAACAGGGTCCA SEQ
ID NO: 61
TRBC2) TGGAIGC T GAIG I TACCCAGACCCCAAGGAATAGGATCACAAAGACAGGAAAGAG
GAT TAT GC T GGAAT GT T C T CAGAC TAAGGGT CAT GATAGAAT GTAC T GGTAT CGA
CAAGACCCAGGACTGGGCCTACGGT T GAT C TAT TAC T CC T T T GAT GT CAAAGATA
TAAACAAAGGAGAGATCTCTGAT GGATACAGT GICTCTCGACAGGCACAGGC TAA
AT TCTCCCTGTCCCTAGAGTCTGCCATCCCCAACCAGACAGCTCT T TACT TCTGT
GCCACCGGCCAGGCGACCCAAGAGACCCAGTACT TCGGGCCAGGCACGCGGCTCC
TGGTGCTCGAGGACCTGAAAAACGTGT TCCCACCCGAGGTCGCTGTGT T TGAGCC
AT CAGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC T GGCCACA
GGCT T C TACCCCGACCACGT GGAGC T GAGC T GGT GGGT GAAT GGGAAGGAGGT GC
ACAGT GGGGT CAGCACAGACCCGCAGCCCC T CAAGGAGCAGCCCGCCC T CAAT GA
CTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCT TCTGGCAGAAC
CCCCGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGCTCTCGGAGAATGACG
AGT GGACCCAGGATAGGGCCAAACC T GT CACCCAGAT CGT CAGCGCCGAGGCC TG
GGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCT
GCCACCAT CC TC TAT GAGATCTTGC TAGGGAAGGCCACCT TGTATGCCGTGCTGG
TCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC
13 (with ATGGCTTCTCTTCTGTTTTTCTGCGGCGCCTTCTACCTGCTCGGCACCG SEQ ID NO:
207
TRBC2) GATCTATGGACGCCGACGTTACCCAGACACCACGGAACAGAATCACCAA
GACCGGCAAGCGGATCATGCTGGAATGCAGCCAGACCAAGGGCCACGAC
CGGATGTACTGGTACAGACAGGATCCAGGACTGGGCCTGAGACTGATCT
ACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGG
CTACAGCGTGTCAAGACAGGCCCAGGCCAAGTTCAGCCTGAGCCTGGAA
AGCGCTATCCCCAACCAGACAGCCCTGTACTTTTGTGCCACCGGCCAGG
CCACACAAGAGACACAGTATTTCGGCCCTGGCACCAGACTGCTGGTGCT
GGAAGATCTGAAGAACGTGTTCCCACCTGAGGTGGCCGTGTTCGAGCCT
TCTGAGGCCGAGATCTCTCACACCCAGAAAGCCACACTCGTGTGTCTGG
CCACCGGCTTCTATCCCGATCACGTGGAACTGTCTTGGTGGGTCAACGG
CAAAGAGGTGCACAGCGGCGTCTGTACCGATCCTCAGCCTCTGAAAGAG
CAGCCCGCTCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAG
TGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGT
GCAGTTCTACGGCCTGAGCGAGAACGATGAGTGGACCCAGGATAGAGCC
AAGCCTGTGACACAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATT
GTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCTGCCACAAT
CCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGTACGCCGTGCTGGTG
TCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGATAGCAGGGGC
Donor Chain Nucleotide sequence SEQ ID NO
HD14 a ATGATGAAATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGG SEQ
ID NO: 62
(TRAV12- TTIGGAGCCAACAGAAGGAGGIGGAGCAGGATCCTGGACCACTCAGTGTICCAGA
3*01) GGGAGCCAT TGT T TCTCTCAACTGCACT TACAGCAACAGTGCT T T TCAATACT
IC
ATGTGGTACAGACAGTAT TCCAGAAAAGGCCCTGAGT T GC T GAT GTACACATAC T
(with
c CAGT GGTAACAAAGAAGAT GGAAGGT T TACAGCACAGGTCGATAAATCCAGCAA
TRAC) GTATATCTCCTTGTTCATCAGAGACTCACAGCCCAGTGATTCAGCCACCTACCTC
TGTGCCTCAGGAGGAGGTGCTCACGGACTCACCT T TGGCAAAGGGACTCATCTAA
71
CA 03117272 2021-04-21
WO 2020/089433 PCT/EP2019/079916
TCATCCAGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTC
TAAATCCAGTGACAAGTCTGTCTGCCTAT TCACCGAT T T TGAT TCTCAAACAAAT
GT GTCACAAAGTAAGGAT TCTGATGTGTATATCACAGACAAAACTGTGCTAGACA
T GAGGTCTATGGACT TCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCT GA
CTT TGCAT GT GCAAACGCCT TCAACAACAGCAT TAT TCCAGAAGACACCT TCT TC
C CCAGCCCAGAAAGT T CC T GT GAT GT CAAGC T GGT CGAGAAAAGC T T T GAAACAG
ATACGAACCTAAACTT TCAAAACCTGTCAGTGATT=TCCGAATCCTCCTCCT
GAAAGTGGCCGGGT T TAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
a AT GAAATCCTTGAGAGT T T TACTAGTGATCCTGTGGCTTCAGT TGAGCTGGGT T T
SEQ ID NO: 192
(TRAV12- GGAGCCAACAGAAGGAGGTGGAGCAGAAT TCTGGACCCCTCAGTGT TCCAGAGGG
2*01) AGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTC
T GGTACAGACAATAT T C T GGGAAAAGCCC T GAGT T GATAAT GT T CATATAC T CCA
(with
AT GGT GACAAAGAAGAT GGAAGGT T TACAGCACAGCTCAATAAAGCCAGCCAGTA
TRAC) T GT T TCTCTGCTCATCAGAGACTCCCAGCCCAGTGAT TCAGCCACCTACCTCTGT
GCC T CAGGAGGAGGT GC T GACGGAC T CACC T T T GGCAAAGGGAC T CAT C TAAT CA
TCCAGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAA
ATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTG
T CACAAAGTAAGGAT T C T GAT GT GTATAT CACAGACAAAAC T GT GC TAGACAT GA
G GT C TAT GGAC T T CAAGAGCAACAGT GC T GT GGCC T GGAGCAACAAAT C T GAC T T
T GCAT GT GCAAACGCCT TCAACAACAGCAT TAT TCCAGAAGACACCT TCTTCCCC
AGCCCAGAAAGT T CC T GT GAT GT CAAGC T GGT CGAGAAAAGC T T T GAAACAGATA
CGAACCTAAACTT TCAAAACCTCTCAGTGATTCGCTTCCGAATCCTCCTCCTGAA
AGIGGCCGGGIT TAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
a ATGATGAAATCCT TGAGAGT T T TACTAGTGATCCTGIGGCTICAGT TGAGCTGGG
SEQ ID NO: 193
(TRAV12- T T TGGAGCCAACAGAAGGAGGIGGAGCAGAAT TCTGGACCCCTCAGIGT TCCAGA
2*02) GGGAGCCAT TGCCTCTCTCAACTGCACT TACAGTGACCGAGGT TCCCAGTCCT TC
T TCTGGTACAGACAATAT TCTGGGAAAAGCCCTGAGT TGATAAT GT CCATATAC T
(with
cc AAT GGTGACAAAGAAGATGGAAGGT T TACAGCACAGCTCAATAAAGCCAGCCA
TRAC) GTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTC
T GT GCC T CAGGAGGAGGT GC T GACGGAC T CACC T T T GGCAAAGGGAC T CAT C TAA
T AT CCAGCCC TATAT CCAGAACCC T GACCC T GCCGT GTACCAGC T GAGAGAC T C
TAAAT CCAGT GACAAGT C T GT C T GCC TAT T CACCGAT T T T GAT T C T CAAACAAAT
GT GT CACAAAGTAAGGAT T C T GAT GT GTATAT CACAGACAAAAC T GT GC TAGACA
T GAGGT C TAT GGAC T T CAAGAGCAACAGT GC T GT GGCC T GGAGCAACAAAT C T GA
CTT TGCAT GT GCAAACGCCT TCAACAACAGCAT TAT TCCAGAAGACACCT TCT TC
C CCAGCCCAGAAAGT T CC T GT GAT GT CAAGC T GGT CGAGAAAAGC T T T GAAACAG
ATACGAACCTAAACTT TCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCT
GAAAGTGGCCGGGT T TAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
a ATGATGAAGTOCCTGOGGGTGCTGCTGGTCATCCTGTGGCTGCAACTGA SEQ ID NO:
208
TRAV12- GCT GGGT CT GGTC CCAGCAGAAAGAGGTGGAACAGGACC CT GGAC CT CT
3*01 WT GTC TGTT CC TGAGGGCGCCAT CGTGTC CC TGAACT GCAC CTACAGCAAC
(with AGC GC CT TC CAGTAC TT CATGTGGTACAGACAGTACAGC CGGAAGGGCC
TRAC) C CGAGCT GC TGAT GTACACATACAGCAGC GGCAACAAAGAGGACGGC CG
GTT TACAGC CCAGGT GGACAAGAGCAGCAAGTACATC TC CC TGTT CATC
C GGGACAGC CAGC CTAGCGATAGCGCCACATAT CT GT GT GCAT CT GGCG
GCGGAGC CGAT GGCC TGACAT TT GGAAAGGGCACC CACC TGAT CATC CA
GCC TTACAT TCAGAACC CC GATC CT GC CGTGTACCAGCT GAGAGACAGC
AAGTCCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGA
C CAAC GT GT CC CAGAGCAAGGACAGCGAC GT GTACAT TACC GATAAGTG
C GT GC TGGACATGCGGAGCAT GGAC TT CAAGAGCAAC TC CGCC GT GGCC
T GGTC CAACAAGAGC GATT TC GC CT GC GC CAAC GC CT TCAACAACAGCA
T TATC CC CGAGGACACATT CT TC CCAAGT CC TGAGAGCAGC TGCGAC GT
GAAGC TGGT GGAAAAGAGC TT CGAGACAGACAC CAAC CT GAAC TT CCAG
AAC CT GAGC GT GATC GGCT TC CGGATC CT GC TGCT GAAAGT GGCC GGCT
T CAAC CT GC TGAT GACC CT GAGACT GT GGTC CAGC
a ATGAAGT CC CT GAGAGT GC TGCT GGTCAT CC TGTGGC TGCAGC TGTC TT
SEQ ID NO: 209
TRAV12- GGGTC TGGT CC CAGCAGAAAGAGGT GGAACAGAACAGCGGC CC TC TGTC
2*01 WT T GT TC CT GAAGGC GC TATC GC CAGC CT GAAC TGCACC TACAGC GATAGA
(with GGCAGCCAGAGCT TC TT CT GGTACAGACAGTACAGCGGCAAGAGC CC CG
TRAC) AGCTGATCATGTTCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTT
TACAGCC CAGC TGAACAAGGC CAGC CAGTAC GT GT CC CT GC TGAT CAGA
GATAGCCAGCC TAGC GACAGC GC CACC TACC TT TGTGCATC TGGT GGCG
GAGCC GATGGC CT GACATT TGGCAAGGGAAC CCAC CT GATCAT CCAGCC
T TACATT CAGAAC CC CGAT CC TGCC GT GTAC CAGC TGAGAGACAGCAAG
AGCAGCGACAAGAGC GT GT GC CT GT TCAC CGAC TT CGACAGCCAGAC CA
ACGTGTC CCAGAGCAAGGACAGC GACGTGTACATCAC CGATAAGT GC GT
GCT GGACAT GC GGAGCATGGACT TCAAGAGCAACAGC GC CGTGGC CT GG
T CCAACAAGAGCGAT TT CGCC TGCGCCAACGCC TT CAACAACAGCAT TA
T CC CC GAGGACACAT TC TT CC CAAGTC CT GAGAGCAGCT GC GACGTGAA
GCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAAC
C TGAGCGTGAT CGGC TT CC GGAT CC TGCT GC TGAAAGTGGC CGGC TT CA
ACC TGCT GATGAC CC TGAGAC TGTGGT CCAGC
72
CA 03117272 2021-04-21
WO 2020/089433 PCT/EP2019/079916
a ATGAAGTCCCTGAGAGTGCTGCTGGTCATCCTGTGGCTGCAGCTGTCTT SEQ ID NO:
210
TRAV12- GGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGAACAGCGGCCCTCTGTC
2*01 mut TGTTCCTGAAGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGATAGA
(with GGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCG
TRAC) AGCTGATCATGTTCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTT
TACAGCCCAGCTGAACAAGGCCAGCCAGTACGTGTCCCTGCTGATCAGA
GATAGCCAGCCTAGCGACAGCGCCACCTACCTTTGTGCATCTGGTGGCG
GAGCCGATGGCCTGACATTTGGCAAGGGAACCCACCTGATCATCCAGCC
TTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGCAAG
AGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACAC
AGGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGT
GCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGG
TCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAACAGCATTA
TCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAA
GCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAAC
CTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGGCTTCA
ACCTGCTGATGACCCTGAGACTGTGGTCCAGC
a ATGATGAAGTCCCTGCGGGTGCTGCTGGTCATCCTGTGGCTGCAACTGA SEQ ID NO:
211
TRAV12- GCTGGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGAACAGCGGCCCTCT
2*02 WT GTCTGTTCCTGAAGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGAT
(with AGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCC
TRAC) CCGAGCTGATCATGAGCATCTACAGCAACGGCGACAAAGAGGACGGCCG
GTTTACAGCCCAGCTGAACAAGGCCAGCCAGTACGTGTCCCTGCTGATC
AGAGATAGCCAGCCTAGCGACAGCGCCACCTACCTTTGTGCATCTGGTG
GCGGAGCCGATGGCCTGACATTTGGCAAGGGAACCCACCTGATCATCCA
GCCTTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGC
AAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGA
CCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTG
CGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCC
TGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAACAGCA
TTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGT
GAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAG
AACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGGCT
TCAACCTGCTGATGACCCTGAGACTGTGGTCCAGC
a ATGATGAAGTCCCTGCGGGTGCTGCTGGTCATCCTGTGGCTGCAACTGA SEQ ID NO:
212
TRAV12- GCTGGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGAACAGCGGCCCTCT
2*02 mut GTCTGTTCCTGAAGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGAT
(with AGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCC
TRAC) CCGAGCTGATCATGAGCATCTACAGCAACGGCGACAAAGAGGACGGCCG
GTTTACAGCCCAGCTGAACAAGGCCAGCCAGTACGTGTCCCTGCTGATC
AGAGATAGCCAGCCTAGCGACAGCGCCACCTACCTTTGTGCATCTGGTG
GCGGAGCCGATGGCCTGACATTTGGCAAGGGAACCCACCTGATCATCCA
GCCTTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGC
AAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGA
CACAGGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTG
CGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCC
TGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAACAGCA
TTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGT
GAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAG
AACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGGCT
TCAACCTGCTGATGACCCTGAGACTGTGGTCCAGC
13 (with ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAG SEQ ID
NO: 63
TRBC1) TGCAT ICI GGAGT CACACAAACCCCAAAGCACCICAICACAGCAACTGGACAGC G
AGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAA
CAGAGCCTGGACCAGGGCCTCCAGT T CC T CAT TCAGTAT TATAATGGAGAAGAGA
GAGCAAAAGGAAACAT TCT TGAACGAT TCTCCGCACAACAGT TCCCTGACT TGCA
CTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCT T TGTAT T TCTGT
GCCAGCGGGAGGGGGGACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAG
TTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATC
AGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC T GGCCACAGGC
T T CT TCCCCGACCACGT GGAGCTGAGCTGGT GGGT GAAT GGGAAGGAGGT GCACA
GT GGGGT CAGCACGGACCCGCAGCCCC T CAAGGAGCAGCCCGCCC T CAAT GAC T C
CAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCT TCTGGCAGAACCCC
CGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGCTCTCGGAGAATGACGAGT
GGACCCAGGATAGGGCCAAACCCGT CACCCAGAT CGT CAGCGCCGAGGCC T GGGG
TAGAGCAGACTGTGGCT T TACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATCCTGTGCTCGT CA
GCGCCCTIGIGTTGATOCCCATGGICAAGAGAAAGGATTIC
13 (with ATGGGCTICAGGCTCCTCTGCTGIGTGGCCTITTGICTCCIGGGAGCAGGCCCAG SEQ ID
NO: 64
TRBC2) TGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCG
73
CA 03117272 2021-04-21
WO 2020/089433 PCT/EP2019/079916
AGTGACGCTGAGATGCTCCCCTAGGICTGGAGACCICTCTGIGTACTGGTACCAA
CAGAGCC T GGACCAGGGCC T CCAGI ICC TCAT T CAGTAT TATAAT GGAGAAGAGA
GAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCA
C TCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCT T TGTAT T TCTGT
GCCAGCGGGAGGGGGGACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAG
TTGTAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATC
AGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC T GGCCACAGGC
TTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACA
GTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTC
CAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCC
CGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGT
GGACCCAGGATAGGGCCAAACC T GT CACCCAGAT CGT CAGCGCCGAGGCC T GGGG
TAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCT TGCTAGGGAAGGCCACCT TGTATGCCGTGCTGGT CA
GTGCCCTCGTGCTGATGGCCATGGICAAGAGAAAGGAT TCCAGAGGC
13 ATGGGTT TTAGAC TGCT GT GC TGCGTGGC CT TC TGTC TGCT TGGAGC TG
SEQ ID NO: 213
TRAV12- GCC CT GT GGATAGCGGC GT TACC CAGACACC TAAGCACC TGAT CACAGC
3*01 WT CACAGGC CAGC GC GT GACC CT GAGATGTT CT CC TAGAAGCGGC GACC TG
(with AGC GT GTAC TGGTAT CAGCAGTC TC TGGACCAGGGCC TGCAGT TC CT GA
TRBC2) T CCAGTACTACAACGGC GAGGAAAGAGCCAAGGGCAACATC CT GGAACG
GTT CAGC GC CCAGCAGT TO CCAGAT CT GCACAGCGAGCT GAAC CT GAGC
AGO CT GGAACT GGGAGATAGC GC CC TGTACT TO TGTGCCAGCGGCAGAG
GCGATAC CGAGGC CT TT TT TGGC CAAGGCAC CAGACT GACC GT GGTGGA
AGATC TGAAGAAC GT GT TO CCAC CT GAGGTGGC CGTGTT CGAGCC TT CT
GAGGC CGAGAT CAGC CACACACAGAAAGC CACACT CGTGTGTC TGGC CA
C CGGC TT CTAT CC CGAT CACGTGGAAC TGTC TT GGTGGGTCAACGGCAA
AGAGGTGCACAGC GGCGTC TGTACC GATC CT CAGC CT CT GAAAGAGCAG
C CC GC TO TGAACGACAGCAGATACT GC CT GT CCAGCAGACT GAGAGT GT
C CGCCAC CT TO TGGCAGAACC CCAGAAAC CACT TCAGAT GC CAGGTGCA
GTTCTACGGCCTGAGCGAGAACGATGAGTGGACCCAGGACAGAGCTAAG
C CC GT GACACAGATC GT GT CT GC C GAAGC TT GGGGCAGAGC C GAT TGTG
GOT TTAC CAGC GAGAGC TACCAGCAGGGC GT GC TGTC TGCCACAATC CT
GTACGAGAT CC TGCT GGGCAAAGCCAC TO TGTACGCC GT GC TGGT GT CT
GCC CT GGTGCT GATGGC CATGGT CAAGCGGAAGGATAGCAGGGGC
Donor Chain Nucleotide sequence SEQ ID NO
HD15 a (with ATGAAATCCITGAGAGITTTACTAGTGATCCTGIGGCTICAGTTGAGCTGGGITT SEQ
ID NO: 65
S4 TRAC) GGAGCCAACAGAAGGAGG I GGAGCAGAA IICT GGACCCCTCAGT G T
TCCAGAGGG
po AGCCAT TGCCTCTCTCAACTGCACT TACAGTGACCGAGGT TCCCAGTCCT TCT T C
pulati
T GGTACAGACAATAT T C T GGGAAAAGCCC T GAGT T GATAAT GT T CATATAC T CCA
on
AT GGT GACAAAGAAGAT GGAAGGT T TACAGCACAGC T CAATAAAGCCAGCCAGTA
TGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGT
GC CGT GATAGGGGGAACTGACAGCTGGGGGAAATTGCAGTTTGGAGCAGGGACCC
AGGT TGTGGTCACCCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAG
AGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAA
ACAAAT GT GT CACAAAGTAAGGAT T C T GAT GT GTATAT CACAGACAAAAC T GT GC
TAGACAT GAGGT C TAT GGAC T T CAAGAGCAACAGT GC T GT GGCC T GGAGCAACAA
AT C T GAC T T T GCAT GT GCAAACGCC T T CAACAACAGCAT TAT T CCAGAAGACAC C
T T CT TCCCCAGCCCAGAAAGT TCCTGTGATGTCAAGCTGGTCGAGAAAAGCT T T G
AAACAGATACGAACCTAAACTT T CAAAACC T GT CAGT GAT TGGGT T CCGAAT C CT
CCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
a (with ATGAAGT CC CT GAGAGT GC TGCT GGTCAT CC TGTGGC TGCAGC TGTC TT
SEQ ID NO: 218
TRAC) GGGTC TGGT CC CAGCAGAAAGAGGT GGAACAGAACAGCGGC CC TC TGTC
T GT TO CT GAAGGC GC TATC GC CAGC CT GAAC TGCACC TACAGC GATAGA
GGCAGCCAGAGCT TO TT CT GGTACAGACAGTACAGCGGCAAGAGC CC CG
AGCTGATCATGTTCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTT
TACAGCC CAGC TGAACAAGGC CAGC CAGTAC GT GT CC CT GC TGAT CAGA
GATAGCCAGCC TAGC GACAGC GC CACC TATC TGTGTGCC GT GATC GGCG
GCACAGATAGC TGGGGCAAAC TC CAGT TT GGCGCT GGCACACAGGTGGT
GGT CACC CC TGACAT TCAGAACC CT GATC CT GC CGTGTACCAGCT GAGA
GACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACA
GCCAGAC CAAC GT GT CC CAGAGCAAGGACAGCGAC GT GTACAT CACC GA
TAAGT GC GT GC TGGACATGCGGAGCAT GGAC TT CAAGAGCAACAGCGCC
GTGGC CT GGTC CAACAAGAGC GATT TO GC CT GC GC CAAC GC CT TCAACA
ACAGCAT TATC CC CGAGGACACATT CT TO CCAAGT CC TGAGAGCAGC TG
C GACGTGAAGC TGGT GGAAAAGAGC TT CGAGACAGACAC CAAC CT GAAC
T TO CAGAAC CT GT CT GT GATC GGCT TO CGGATC CT GC TGCT GAAGGT GG
C CGGC TT CAAT CT GC TGAT GACC CT GAGACT GT GGTC CAGC
13 (with ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCGGGTGAGT SEQ
ID NO: 66
TRBC1) TGGTCCCCATGGAAAC GGGAG 1 TACGCAGACACCAAGACACC T GGT CAT
GGGAAT
GACAAATAAGAAGTCTT T GAAAT GT GAACAACAT C T GGG T CATAACGC TATGTAT
TGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTCATGTTTGICTACAGICTIG
74
CA 03117272 2021-04-21
WO 2020/089433 PCT/EP2019/079916
AA AA
TGAAAACAACAGTGTGCCAAGTCGCT TCTCACCTGAATGCCCCAA
CAGC ICICAC I TAT TCCT TCACCTACACACCCTGCAGCCAGAAGACTCGGCCCT G
TATCTCTGCGCCAGCAGCCAAGAAGAGGGGGCTGTCTATGGCTACACCTTCGGTT
C GGGGACCAGGT TAACCGT T GTAGAGGACC T GAACAAGGT GT T CCCACCCGAGG T
C GC TGT GT T T GAGCCAT CAGAAGCAGAGAT C TCCCACACCCAAAAGGCCACAC T G
G TGTGCCTGGCCACAGGCT TCT TCCCCGACCACGTGGAGCTGAGCTGGTGGGTGA
AT GGGAAGGAGGT GCACAGT GGGGT CAGCACGGACCCGCAGCCCC T CAAGGAGCA
GCCCGCCCT CAAT GAC T CCAGATAC T GCC T GAGCAGCCGCC T GAGGGT CT CGGCC
ACCT TCTGGCAGAACCCCCGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGC
T C T CGGAGAAT GACGAGT GGACCCAGGATAGGGCCAAACCCGT CACCCAGAT CG T
CAGCGCCGAGGCC T GGGGTAGAGCAGAC T GT GGC T T TACC T CGGT GT CC TACCAG
CAAGGGGT CC T GT C T GCCACCAT CC T C TAT GAGAT CC T GC TAGGGAAGGCCACCC
T GTAT GOT GT GOT GGTCAGCGCCCT T GT GT T GATGGCCAT GGT CAAGAGAAAGGA
TTTC
13 (with ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCGGGTGAGT SEQ
ID NO: 67
TRBC2) T GGI CCCCAT GGAAACGGGAG I TACGCAGACACCAAGACACC T GGT CAT
GGCAAT
GACAAATAAGAAGTCT T TGAAAT GT GAACAACATCTGGGT CATAACGC TAT GTAT
T GGTACAAGCAAAGT GC TAAGAAGCCACTGGAGCTCAT GT T TGTCTACAGTCT T G
AAGAACGGGT TGAAAACAACAGT GT GCCAAGT CGCT TCTCACCTGAAT GCCCCAA
CAGC T C T CAC T TAT T CC T T CACC TACACACCC T GCAGCCAGAAGAC T CGGCCC T G
TATCTCTGCGCCAGCAGCCAAGAAGAGGGGGCTGTCTATGGCTACACCTTCGGTT
C GGGGACCAGGT TAACCGT T GTAGAGGACC T GAAAAACGT GT T CCCACCCGAGG T
C GC T GT GT T T GAGCCAT CAGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T G
G TGTGCCTGGCCACAGGCT TCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGA
AT GGGAAGGAGGT GCACAGT GGGGT CAGCACAGACCCGCAGCCCC T CAAGGAGCA
GCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCC
ACCT TCTGGCAGAACCCCCGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGC
TC T CGGAGAAT GACGAGT GGACCCAGGATAGGGCCAAACC T GT CACCCAGAT CG T
CAGCGCCGAGGCC T GGGGTAGAGCAGAC T GT GGC T T CACC T CCGAGT C T TACCAG
CAAGGGGT CC T GT C T GCCACCAT CC T C TAT GAGAT C T T GC TAGGGAAGGCCACC T
TGTATGCCGTCCTCCTCAGTCCCCTCCTCCTGATCCCCATCCTCAACAGAAAGGA
TTCCAGAGGC
13 (with ATGGGATGTAGACTTCTGTGTTGCGCCGTGCTGTGTCTGCTTGGAGCTG SEQ ID NO:
219
TRBC2) GCGAACT GGTGCC TATGGAAACC GGCGTGAC CCAGACAC CTAGACAC CT
GGT CATGGGCATGACAAACAAGAAAAGCC TGAAGT GC GAGCAGCACC TG
GGC CACAAT GC CATGTACT GGTACAAGCAGAGC GC CAAGAAAC CC CT GG
AAC TGAT GT TC GT GTACAGCC TGGAAGAGAGGGTC GAGAACAACAGC GT
GCC CAGCAGAT TCAGCC CT GAGT GC CC TAATAGCAGC CACC TGT T TC TG
CAT CT GCACAC CC TGCAGC CT GAGGAC TC TGCC CT GTAT CT GT GT GC CA
GCAGC CAAGAGGAAGGC GC CGT T TACGGC TACACAT T TGGCAGCGGCAC
CAGAC TGAC CGTGGT GGAAGATC TGAAGAAC GT GT TC CCAC CT GAGGTG
GCC GT GT TC GAGC CT TC TGAGGC CGAGAT CAGC CACACACAGAAAGC CA
CAC TC GT GT GT CT GGCCAC CGGC T T CTAT CC CGAT CACGTGGAAC TGTC
T TGGT GGGT CAAC GGCAAAGAGGTGCACAGC GGCGTC TGTACC GATC CT
CAGCC TC TGAAAGAGCAGC CC GC TC TGAACGACAGCAGATACT GC CT GA
GCAGCAGAC TGAGAGTGTC CGCCAC CT TC TGGCAGAACC CCAGAAAC CA
CTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGATGAGTGG
ACC CAGGATAGAGCCAAGC CT GT GACACAGATC GT GT CT GC CGAAGC CT
GGGGCAGAGCC GAT T GT GGCT T TAC CAGC GAGAGC TACCAGCAGGGC GT
GCT GT CT GC CACAAT CC TGTACGAGAT CC TGCT GGGAAAAGCCAC TC TG
TAC GC TGTGCT GGTGTC CGCT CT GGTGCT GATGGC CATGGT CAAGCGGA
AGGATAGCAGGGGC
HD15 a (with ATGATATCCITGAGAGITTTACTGGTGATCCTGIGGCTICAGTTAAGCTGGGITT SEQ
ID NO: 68
S1 IFNg TRAC) GGAGCCAACGGAAGGAGG I GGAGCAGGAT CC
IGGACCCTICAAIGIICCAGAGGG
enriche AGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCTCAGTCTTTCTTC
T d GGTACAGACAGGAT T
GCAGGAAAGAACC TAAGT T GC T GAT GT CCGTATAC T CCA
GTGGTAATGAAGATGGAAGGT TTACAGCACAGCTCAATAGAGCCAGCCAGTATAT
populati TTCCCTGCTCATCAGAGACTCCAAGCTCAGTGATTCAGCCACCTACCTCTGTGTG
on GTGCCCCGGGGGCTTTCAACTGACAGCTGGGGGAAATTGCAGTTTGGAGCAGGGA
CCCAGGT TGTGGTCACCCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGC T
GAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCT
CAAACAAAT GT GT CACAAAGTAAGGAT T C T GAT GT GTATAT CACAGACAAAAC TG
T GC TAGACAT GAGGT C TAT GGAC T T CAAGAGCAACAGT GC T GT GGCC T GGAGCAA
CAAATCTGACT T T GCAT GT GCAAACGCC T T CAACAACAGCAT TAT TCCAGAAGAC
ACCT TCT TCCCCAGCCCAGAAAGT TCCTGTGATGTCAAGCTGGTCGAGAAAAGC T
T TGAAACAGATACGAACCTAAACTT T CAAAACC T GT CAGT GAT T GGGT T CCGAAT
CC TCCTCCTGAAAGTGGCCGGGT T T AATCTGCTCATGACGCTGCGGCTGTGGT CC
AGC
a (with ATGATCAGCCTGAGAGTGCTGCTGGTCATCCTGTGGCTGCAGCTGTCTT SEQ ID NO:
220
TRAC) GGGTC TGGT CC CAGC GGAAAGAGGT GGAACAGGAC CC CGGACC T T TCAA
T GT GC CT GAAGGC GC CACC GT GGCC TI CAAC TGCACC TACAGCAATAGC
GCCAGCCAGAGCT TC T T CT GGTACAGACAGGAC TGCC GGAAAGAACC CA
AGCTGCTGATGAGCGTGTACAGCAGCGGCAACGAGGACGGCAGATTCAC
CA 03117272 2021-04-21
WO 2020/089433 PCT/EP2019/079916
AGCCCAGCTGAACAGAGCCAGCCAGTACATCAGCCTGCTGATCCGGGAT
AGCAAGCTGAGCGATAGCGCCACCTACCTGTGCGTGGTGCCTAGAGGCC
TGAGCACAGATTCTTGGGGCAAGCTGCAGTTCGGAGCCGGAACACAGGT
GGTGGTCACCCCTGACATTCAGAACCCTGATCCTGCCGTGTACCAGCTG
AGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCG
ACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCAC
CGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGC
GCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCA
ACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAG
CTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTG
AACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAG
TGGCCGGCTTCAACCTGCTCATGACCCTGAGACTGTGGTCCAGC
13 (with ATGGGICCIGGGCTICTCCACIGGATGGCCCITTGICTCCTIGGAACAGGICATG SEQ
ID NO: 69
TRBC1) GGGATGCCAT GGICATCCAGAACCCAAGATACCAGGITACCCAGITIGGAAAGCC
AGTGACCCTGAGT TGT TCTCAGACT T TGAACCATAACGTCATGTACTGGTACCAG
CAGAAGT CAAGT CAGGCCCCAAAGC T GC T GT T CCAC TAC TAT GACAAAGAT TTTA
ACAATGAAGCAGACACCCCTGATAACT TCCAATCCAGGAGGCCGAACACT TCTIT
CTGCT T TCT TGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGT
GCCACCAGCAGGGAGGGGCTAGCGGCAGATACGCAGTAT T T TGGCCCAGGCACCC
GGCTGACAGTGCTCGAGGACCTGAACAAGGTGT TCCCACCCGAGGTCGCTGTGT T
T GAGCCAT CAGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC TG
GCCACAGGCT TCT TCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG
AGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCT TCTGG
CAGAACCCCCGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGCTCTCGGAGA
AT GACGAGT GGACCCAGGATAGGGCCAAACCCGT CACCCAGAT CGT CAGCGCCGA
GGCC T GGGGTAGAGCAGAC T GI GGC T T TACC T CGGT GI CC TACCAGCAAGGGGT C
C T GTCTGCCACCATCCTCTAT CACATCCTGCTAGGGAAGGCCACCCTGTAT CC T G
TGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
13 (with ATGGGICCTGGGCTICTCCACIGGATGGCCCITTGICTCCTTGGAACAGGICATG SEQ
ID NO: 70
TRBC2) GGGATGCCATGGICATCCAGAACCCAAGATACCAGGTIACCCAGTITGGAAAGCC
AGTGACCCTGAGT TGT TCTCAGACT T TGAACCATAACGTCATGTACTGGTACCAG
CAGAAGT CAAGT CAGGCCCCAAAGC T GC T GT T CCAC TAC TAT GACAAAGAT T T TA
ACAATGAAGCAGACACCCCTGATAACT TCCAATCCAGGAGGCCGAACACT TCTIT
CTGCT T TCT TGACATCCGCTCACCAGGCCIGGGGGACGCAGCCATGTACCTGIGT
GCCACCAGCAGGGAGGGGCTAGCGGCAGATACGCAGTAT T T TGGCCCAGGCACCC
GGCTGACAGTGCTCGAGGACCTGAAAAACGTGT TCCCACCCGAGGTCGCTGTGT T
T GAGCCAT CAGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC TG
GCCACAGGCT TCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG
AGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGG
CAGAACCCCCGCAACCACTICCGCTGTCAAGTCCAGTICTACGGGCTCTCGGAGA
AT GACGAGT GGACCCAGGATAGGGCCAAACC T GT CACCCAGAT CGT CAGCGCCGA
GGCC T GGGGTAGAGCAGAC T GT GGC T TCACCTCCGAGTCT TACCAGCAAGGGGTC
CTGTCTGCCACCATCCTCTATGAGATCT TGCTAGGGAAGGCCACCT TGTATGCCG
TGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGAT TCCAGAGG
C
13 (with ATGGGACCTGGACTTCTTCATTGGATGGCCCTGTGTCTGCTCGGCACAG SEQ ID NO:
221
TRBC2) GACATGGCGACGCTATGGTCATTCAGAACCCCAGATACCAAGTGACCCA
GTTCGGCAAGCCCGTGACACTGAGCTGTAGCCAGACACTGAACCACAAC
GTGATGTACTGGTATCAGCAGAAGTCCTCTCAGGCCCCTAAGCTGCTGT
TCCACTACTACGACAAGGACTTCAACAACGAGGCCGACACACCCGACAA
CTTCCAGAGCAGAAGGCCCAATACCAGCTTCTGCTTCCTGGACATCAGA
AGCCCTGGCCTGGGAGATGCCGCCATGTATCTGTGTGCCACCAGCAGAG
AAGGCCTGGCCGCCGATACACAGTATTTCGGCCCTGGCACCAGACTGAC
CGTGCTCGAGGATCTGAAGAACGTGTTCCCACCTGAGGTGGCCGTGTTC
GAGCCTTCTGAGGCCGAGATCAGCCACACACAGAAAGCCACACTCGTGT
GTCTGGCCACCGGCTTCTATCCCGATCACGTGGAACTGTCTTGGTGGGT
CAACGGCAAAGAGGTGCACAGCGGCGTCTGTACCGATCCTCAGCCTCTG
AAAGAGCAGCCCGCTCTGAACGACAGCAGATACTGCCTGAGCAGCAGAC
TGAGAGTGTCCGCCACCTTCTGGCAGAACCCTCGGAACCACTTCAGATG
CCAGGTGCAGTTCTACGGCCTGAGCGAGAACGATGAGTGGACCCAGGAT
AGAGCCAAGCCTGTGACTCAGATCGTGTCTGCCGAAGCCTGGGGCAGAG
CCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCTGC
CACAATCCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGTACGCCGTG
CTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGATAGCA
GGGGC
Donor Chain Nucleotide sequence SEQ ID NO
76
CA 03117272 2021-04-21
WO 2020/089433 PCT/EP2019/079916
Patient a (with ATGGCTTTGCAGAGCACTCTGGGGGCGGTGTGGCTAGGGCTTCTCCTCAACTCTC
SEC) ID NO: 162
1 TRAC) TCTGGAAGGTTGCAGAAAGCAAGGACCAAGTGTTTCAGCCTTCCACAGTGGCATC
TTCAGAGGGAGCTGTGGTGGAAATCTTCTGTAATCACTCTGTGTCCAATGCTTAC
AACTTCTTCTGGTACCTTCACTTCCCGGGATGTGCACCAAGACTCCTTGTTAAAG
dI rect
GCTCAAAGCCTTCTCAGCAGGGACGATACAACATGACCTATGAACGGTTCTCTTC
sequenc ATCGCTGCTCATCCTCCAGGTGCGGGAGGCAGATGCTGCTGTTTACTACTGTGCT
ing GCCCCTAACGACTACAAGCTCAGCTTTGGAGCCGGAACCACAGTAACTGTAAGAG
upon CAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAG
sorting TGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAA
AGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTA
TGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATG
TGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCA
GAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACC
TAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGC
CGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
pl (with ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACG SEQ
ID NO: 163
TRBC1) CAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAA
TGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGA
CAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAAC
TAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATC
TTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTC
TGTGCCAGCAGCAGCGGACTAGCGTTCTACGAGCAGTACTTCGGGCCGGGCACCA
GGCTCACGGTCACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTT
TGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTG
GCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG
AGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGG
CAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGA
ATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGA
GGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTC
CTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTG
TGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
pf (with ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACG SEC)
ID NO: 164
TRBC2) CAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAA
TGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGA
CAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAAC
TAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATC
TTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTC
TGTGCCAGCAGCAGCGGACTAGCGTTCTACGAGCAGTACTTCGGGCCGGGCACCA
GGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTT
TGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTG
GCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG
AGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGG
CAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGA
ATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGA
GGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTC
CTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCG
TGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGG
132 (with ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACA SEQ ID NO:
165
TRBC1) TAGAAGCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGAC
TGTGACTCTGAGATGTGACCCAATTTCTGGACATGATAATCTTTATTGGTATCGA
CGTGTTATGGGAAAAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAAC
AGGATGAGTCCGGTATGCCCAACAATCGATTCTTAGCTGAAAGGACTGGAGGGAC
GTATTCTACTCTGAAGGTGCAGCCTGCAGAACTGGAGGATTCTGGAGTTTATTTC
TGTGCCAGCAGCCAATTGTCAGGGCGCGACTCCTACGAGCAGTACTTCGGGCCGG
GCACCAGGCTCACGGTCACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGC
TGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTG
TGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATG
GGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCC
CGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACC
TTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCT
CGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAG
CGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAA
GGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGT
ATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTT
p2 (with ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACA SEQ
ID NO: 166
TRBC2) TAGAAGCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGAC
TGTGACTCTGAGATGTGACCCAATTTCTGGACATGATAATCTTTATTGGTATCGA
CGTGTTATGGGAAAAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAAC
AGGATGAGTCCGGTATGCCCAACAATCGATTCTTAGCTGAAAGGACTGGAGGGAC
GTATTCTACTCTGAAGGTGCAGCCTGCAGAACTGGAGGATTCTGGAGTTTATTTC
TGTGCCAGCAGCCAATTGTCAGGGCGCGACTCCTACGAGCAGTACTTCGGGCCGG
GCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGC
77
CA 03117272 2021-04-21
WO 2020/089433 PCT/EP2019/079916
TGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTG
TGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATG
GGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCC
CGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACC
TTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCT
CGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAG
CGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAA
GGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGT
ATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTC
Donor Chain Nucleotide sequence õõõõõõõõõõõ am ID NO
Patient al (with .:GATGCTTGCC,
ACATTGA, SEQ ID NO. 167
1 TRAC) GAGCTCAGTCAGTGGCTCAGCCGGAAGATCAGGTCAACGTTGCTGAAGGGAATCC
TCTGACTGTGAAATGCACCTATTCAGTCTCTGGAAACCCTTATCTTTTTTGGTAT
GTTCAATACCCCAACCGAGGCCTCCAGTTCCTTCTGAAATACATCACAGGGGATA
growing
ACCTGGTTAAAGGCAGCTATGGCTTTGAAGCTGAATTTAACAAGAGCCAAACCTC
colony CTTCCACCTGAAGAAACCATCTGCCCTTGTGAGCGACTCCGCTTTGTACTTCTGT
GCTGTGAGAGACGGTGGTGCTACAAACAAGCTCATCTTTGGAACTGGCACTCTGC
TTGCTGTCCAGCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGA
CTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACA
AATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAG
ACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATC
TGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTC
TTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAA
CAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCT
CCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
a2 (with ATGAGGCAAGTGGCGAGAGTGATCGTGTTccTGAcccTGAGTAcTTTGAGCCTTG SEQ ID NO 168
TRAC) CTAAGACCACCCAGCCCATCTCCATGGACTCATATGAAGGACAAGAAGTGAACAT
AACCTGTAGCCACAACAACATTGCTACAAATGATTATATCACGTGGTACCAACAG
TTTCCCAGCCAAGGACCACGATTTATTATTCAAGGATACAAGACAAAAGTTACAA
ACGAAGTGGCCTCCCTGTTTATCCCTGCCGACAGAAAGTCCAGCACTCTGAGCCT
GCCCCGGGTTTCCCTGAGCGACACTGCTGTGTACTACTGCCTCGTGGGTGGTTAT
ACTGGAGGCTTCAAAACTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCAA
ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGA
CAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGT
AAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGG
ACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGC
AAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAA
AGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAA
ACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGG
GTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
p
(with ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCC SEQ ID NO 169
TRBC1) TGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAA
GTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGA
CAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGA
CTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAA
TTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGT
GCCAGCAGTACGCTTGGGGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCG
TACTGGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATC
AGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGC
TTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACA
GTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTC
CAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCC
CGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGT
GGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG
TAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCA
GCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
p
(with ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCC SEQ ID NO: 170
TRBC2) TGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAA
GTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGA
CAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGA
CTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAA
TTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGT
GCCAGCAGTACGCTTGGGGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCG
TACTGGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATC
AGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGC
TTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACA
GTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTC
CAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCC
CGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGT
GGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG
TAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCA
GTG:rrrTrGTG:rTGATGGrrATGGTCAAGAGAAAGGATTCCAGAGGC
Donor Chain Nucleotide sequence sea __ ID No
78
CA 03117272 2021-04-21
WO 2020/089433 PCT/EP2019/079916
Patient a 1 (with ATGCTCCTGCTGCTCGTCCCAGCGTTccAGGTGATTTTTAcccTGGGAGGAACCA
SEQ ID NO: 171
2 TRAC) GAGCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCC
TGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTGTATCTCTTCTGGTAT
GTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTATTTATCAGGATCCA
CCCTGGTTGAAAGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTC
CTTCCACTTGAGGAAACCCTCAGTCCATATAAGCGACACGGCTGAGTACTTCTGT
GCTGTGACCCTGCTTTCGATTGAGCCTTCGGCTGGGGGTTACCAGAAAGTTACCT
TTGGAATTGGAACAAAGCTCCAAGTCATCCCAAATATCCAGAACCCTGACCCTGC
CGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACC
GATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCA
CAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGT
GGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATT
ATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGG
TCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGAT
TGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTG
CGGCTGTGGTCCAGC
a 2 (with ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGA SEQ ID NO:
172
TRAC) ATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAG
CGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACTACTTCCCTTGGTAT
AAGCAAGAACTTGGAAAAAGACCTCAGCTTATTATAGACATTCGTTCAAATGTGG
GCGAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAACATTT
CTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGTGCA
GCAACCTCCCGCGATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAAC
CAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAG
TGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAA
AGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTA
TGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATG
TGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCA
GAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACC
TAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGC
CGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
13 1 (with ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACG SEQ ID NO:
173
TRBC1) CAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAA
TGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGA
CAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAAC
TAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATC
TTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTC
TGTGCCAGCAGCTTAGAAGGAAGGGCCATGCCCAGGGACAGCCACCAAGAGACCC
AGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAACAAGGTGTT
CCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAA
AAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGA
GCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCC
CCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTG
AGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCC
AGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGT
CACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCG
GTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAG
GGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGT
CAAGAGAAAGGATTTC
p 1 (with ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACG SEC) ID NO:
174
TRBC2) CAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAA
TGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGA
CAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAAC
TAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATC
TTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTC
TGTGCCAGCAGCTTAGAAGGAAGGGCCATGCCCAGGGACAGCCACCAAGAGACCC
AGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTT
CCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAA
AAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGA
GCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCC
CCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTG
AGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCC
AGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGT
CACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCC
GAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAG
GGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGT
CAAGAGAAAGGATTCCAGAGGC
p 2 (with ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATG SEQ ID NO:
175
TRBC1) GGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCC
AGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAG
CAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTA
ACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGAACACTTCTTT
CTGCTTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGT
GCCACCAGCTGGGGGCTAAACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGG
TCACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATC
AGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGC
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TTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACA
GTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTC
CAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCC
CGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGT
GGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG
TAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCA
GCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
I p 2 (with ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATG SEQ ID NO
176
TRBC2) GGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCC
AGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAG
CAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTA
ACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGAACACTTCTTT
CTGCTTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGT
GCCACCAGCTGGGGGCTAAACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGG
TCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATC
AGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGC
TTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACA
GTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTC
CAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCC
CGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGT
GGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG
TAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCA
GTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC
Donor Chain õ õõõ, SEQ ID NO
Patient a 1 (with ATGcTGAcTGccAGccTGTTGAGGGcAGTcATAGccrccATcTGTGTTGTATccA
SEQ ID NO 177
3 TRAC) GCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGA
GGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATACTACTTATTACTTATTC
TGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTT
TTGATGAGCAAAATGAAATAAGTGGTCGGTATTCTTGGAACTTCCAGAAATCCAC
CAGTTCCTTCAACTTCACCATCACAGCCTCACAAGTCGTGGACTCAGCAGTATAC
TTCTGTGCTCTGCCCGACAAGGTGATATTTGGGCCAGGGACAAGCTTATCAGTCA
TTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATC
CAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCA
CAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGT
CTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGC
ATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGC
CCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGA
ACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGT
GGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
Ia 2 (with ATGCTCCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTG SEQ ID NO
178
TRAC) GTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGT
CTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAG
GAACCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGA
CCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCT
GAATATCTCAGCATCCATACCTAGTGATGTAGGCATCTACTTCTGTGCTGGGCTA
TATGCTACAAACAAGCTCATCTTTGGAACTGGCACTCTGCTTGCTGTCCAGCCAA
ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGA
CAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGT
AAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGG
ACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGC
AAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAA
AGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAA
ACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGG
GTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC
13 (with ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAG SEQ
ID NO 179
TRBC1) TGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCG
AGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAA
CAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGA
GAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCA
CTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGT
GCCAGCAGCGTATCGGCAGGGAGCACCGGGGAGCTGTTTTTTGGAGAAGGCTCTA
GGCTGACCGTACTGGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTT
TGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTG
GCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG
AGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGG
CAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGA
ATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGA
GGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTC
CTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTG
TGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
p (with ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAG SEQ
ID NO 180
TRBC2) TGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCG
AGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAA
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CAGAGCCTGGACCAGGGCCTCCAGT TCCTCAT TCAGTAT TATAATGGAGAAGAGA
GAGCAAAAGGAAACAT TC 1 TGAACGAT IC ICCGCACAAGAGT TCCC TGAC 1 I GCA
CTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCT T TGT AT TTCTGT
GCCAGCAGCGTATCGGCAGGGAGCACCGGGGAGCTGT TTTT TGGAGAAGGC T C TA
GGCTGACCGTACTGGAGGACCTGAAAAACGTGT TCCCACCCGAGGTCGCTGTGT T
T GAGCCAT CAGAAGCAGAGAT C T CCCACACCCAAAAGGCCACAC T GGT GT GCC TG
GCCACAGGCT TCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG
AGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCT TCTGG
CAGAACCCCCGCAACCACT TCCGCTGTCAAGTCCAGT TCTACGGGCTCTCGGAGA
AT GACGAGT GGACCCAGGATAGGGCCAAACC T GT CACCCAGAT CGT CAGCGCCGA
GGCC T GGGGTAGAGCAGAC T GT GGC T TCACCTCCGAGTCT TACCAGCAAGGGGTC
CTGTCTGCCACCATCCTCTATGAGATCT TGCTAGGGAAGGCCACCT TGTATGCCG
TGC T GGT CAGT GCCC T CGT GC T GAT GGCCAT GGT CAAGAGAAAGGAT TCCAGAGG
C
Accordingly, the invention provides an isolated polynucleotide comprising one
or more
nucleotide sequences selected from the group consisting of SEQ ID NOs: 56-70,
162-180,
192, 193, 204-213 and 218-221, or variants thereof having at least 40%, at
least 50%, at
least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence identity
thereto.
The invention also provides a TCR comprising an a chain encoded by a
nucleotide
sequence selected from the group consisting of SEQ ID NOs: 56, 59, 62, 65, 68,
162, 167,
168, 171, 172, 177, 178, 192, 193, 204, 206, 208-212, 218 and 220, and
variants thereof
having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%,
at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at least
99% sequence identity thereto.
The invention also provides a TCR comprising a 13 chain encoded by a
nucleotide sequence
selected from the group consisting of SEQ ID Nos: 57, 58, 60, 61, 63, 64, 66,
67, 69, 70,
163, 164, 165, 166, 169, 170, 173, 174, 175, 176, 179, 180, 205, 207, 213, 219
and 221,
.. and variants thereof having at least 40%, at least 50%, at least 60%, at
least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at
least 98%, or at least 99% sequence identity thereto.
Further provided by the invention are isolated polynucleotide sequences
derived from the
sequences present in Table 2. For example, the present invention provides an
isolated
.. polynucleotide encoding a variable region of a TCR according to the
invention, wherein the
isolated polynucleotide comprises a stretch of nucleotides of any one of SEQ
ID Nos: 56-70,
162-180, 192, 193, 204-213 and 218-221.
The variant sequences may have additions, deletions or substitutions, of one
or more bases.
If the variation involves addition(s) or deletion(s) they may either occur in
threes or be
balanced (i.e. an addition for each deletion) so that the variation does not
cause a frame-
shift for translation of the remainder of the sequence.
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Some or all of the variations may be "silent" in the sense that they do not
affect the
sequence of the encoded protein due to the degeneracy of the genetic code.
Some or all of the variations may produce conservative amino acid
substitutions, additions or
deletions as explained above. The variation may be concentrated in one or more
regions,
.. such as the regions encoding the constant regions, the linker, or the
framework regions of
the a or 13 chains, or they may be spread throughout the molecule.
The variant sequence should retain the capacity to encode all or part of a TCR
amino acid
sequence which binds to a WT1 peptide.
Codon optimisation
The polynucleotides used in the present invention may be codon-optimised.
Codon
optimisation has previously been described in WO 1999/41397 and WO 2001/79518.
Different cells differ in their usage of particular codons. This codon bias
corresponds to a
bias in the relative abundance of particular tRNAs in the cell type. By
altering the codons in
the sequence so that they are tailored to match with the relative abundance of
corresponding
tRNAs, it is possible to increase expression. By the same token, it is
possible to decrease
expression by deliberately choosing codons for which the corresponding tRNAs
are known
to be rare in the particular cell type. Thus, an additional degree of
translational control is
available.
Many viruses, including HIV and other lentiviruses, use a large number of rare
codons and
by changing these to correspond to commonly used mammalian codons, increased
expression of the packaging components in mammalian producer cells can be
achieved.
Codon usage tables are known in the art for mammalian cells, as well as for a
variety of
other organisms.
Codon optimisation may also involve the removal of mRNA instability motifs and
cryptic
splice sites.
Vector
The invention provides a vector comprising a polynucleotide described herein.
A vector is a tool that allows or facilitates the transfer of an entity from
one environment to
another. In accordance with the invention, and by way of example, some vectors
used in
recombinant nucleic acid techniques allow entities, such as a segment of
nucleic acid (e.g. a
heterologous DNA segment, such as a heterologous Cdna segment), to be
transferred into a
target cell. The vector may serve the purpose of maintaining the heterologous
nucleic acid
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(DNA or RNA) within the cell, facilitating the replication of the vector
comprising a segment
of nucleic acid, or facilitating the expression of the protein encoded by a
segment of nucleic
acid. Vectors may be non-viral or viral. Examples of vectors used in
recombinant nucleic
acid techniques include, but are not limited to, plasmids, chromosomes,
artificial
chromosomes and viruses. The vector may be single stranded or double stranded.
It may
be linear and optionally the vector comprises one or more homology arms. The
vector may
also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form,
the vector may
itself be a nucleotide of interest.
The vectors used in the invention may be, for example, plasmid or virus
vectors and may
include a promoter for the expression of a polynucleotide and optionally a
regulator of the
promoter.
Vectors comprising polynucleotides used in the invention may be introduced
into cells using
a variety of techniques known in the art, such as transformation, transfection
and
transduction. Several techniques are known in the art, for example
transduction with
recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-
associated viral,
baculoviral and herpes simplex viral vectors, Sleeping Beauty vectors; direct
injection of
nucleic acids and biolistic transformation.
Non-viral delivery systems include but are not limited to DNA transfection
methods. Here,
transfection includes a process using a non-viral vector to deliver a gene to
a target cell.
Typical transfection methods include electroporation, DNA biolistics, lipid-
mediated
transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes,
lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles
(CFAs) (Nature
Biotechnology 1996 14; 556) and combinations thereof.
The term "transfection" is to be understood as encompassing the delivery of
polynucleotides
to cells by both viral and non-viral delivery.
In addition, the invention may employ gene targeting protocols, for example
the delivery of
DNA-modifying agents.
The term "vector" includes an expression vector i.e. a construct capable of in
vivo or in vitrolex
vivo expression. Expression may be controlled by a vector sequence, or, for
example in the
case of insertion at a target site, expression may be controlled by a target
sequence. A vector
may be integrated or tethered to the cell's DNA.
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Viral delivery systems include but are not limited to adenovirus vector, an
adeno-associated
viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral
vector, and a
baculoviral vector.
Retroviruses are RNA viruses with a life cycle different to that of lytic
viruses. In this regard,
a retrovirus is an infectious entity that replicates through a DNA
intermediate. When a
retrovirus infects a cell, its genome is converted to a DNA form by a reverse
transcriptase
enzyme. The DNA copy serves as a template for the production of new RNA
genomes and
virally encoded proteins necessary for the assembly of infectious viral
particles.
There are many retroviruses, for example murine leukemia virus (MLV), human
immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse
mammary
tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV),
Moloney
murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),
Moloney
murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian
myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV) and
all other
retroviridiae including lentiviruses.
A detailed list of retroviruses may be found in Coffin eta! ("Retroviruses"
1997 Cold Spring
Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).
Lentiviruses also belong to the retrovirus family, but they can infect both
dividing and non-
dividing cells (Lewis et al (1992) EMBO J. 3053-3058).
The vector may be capable of transferring a nucleotide sequence encoding a WT1-
specific
TCR described herein to a cell, such as a T-cell, such that the cell expresses
the WT1-
specific TCR. Preferably the vector will be capable of sustained high-level
expression in T-
cells, so that the introduced TCR may compete successfully with the endogenous
TCR for a
limited pool of CD3 molecules.
Increasing the supply of CD3 molecules may increase TCR expression, for
example, in a cell
that has been modified to express the TCRs of the invention. Accordingly, the
vector of the
invention may further comprise one or more genes encoding CD3-gamma, CD3-
delta, CD3-
epsilon and/or CD3-zeta. In one embodiment, the vector of the invention
comprises a gene
encoding CD3-zeta. The vector may comprise a gene encoding CD8. The vector may
encode a selectable marker or a suicide gene, to increase the safety profile
of the genetically
engineered cell, e.g. a cell of the invention, or a cell that has been
modified to express the
TCRs of the invention (Bonini, Science 1997, Ciceri, Bonini Lancet Oncol.
2009, Oliveira et
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al., STM 2015). The genes comprised in the vector of the invention may be
linked by self-
cleaving sequences, such as the 2A self-cleaving sequence.
Alternatively one or more separate vectors encoding a CD3 gene may be provided
for co-
transfer to a cell simultaneously, sequentially or separately with one or more
vectors of the
invention, e.g. one or more vectors encoding TCRs of the invention.
Cell
The invention relates to a cell comprising a polynucleotide or a vector
according to the
invention.
The cell may be a T-cell, a lymphocyte, or a stem cell. The T-cell, the
lymphocyte, or the
stem cell may be selected from the group consisting of CD4 cells, CD8 cells,
naive T-cells,
memory stem T-cells, central memory T-cells, double negative T-cells, effector
memory T-
cells, effector T-cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2
cells, Tc2 cells, Th17
cells, Th22 cells, gamma/delta T-cells, natural killer (NK) cells, natural
killer T (NKT) cells,
cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent
stem cells.
The type of cell may be selected in order to provide desirable and
advantageous in vivo
persistence and to provide desirable and advantageous functions and
characteristics to the
cells of invention.
The cell may have been isolated from a subject.
The cell of the invention may be provided for use in adoptive cell transfer.
As used herein
the term "adoptive cell transfer" refers to the administration of a cell
population to a patient.
Typically, the cells are T-cells isolated from a subject and then genetically
modified and
cultured in vitro in order to express a TCR of the invention before being
administered to the
patient.
Adoptive cell transfer may be allogenic or autologous.
By "autologous cell transfer" it is to be understood that the starting
population of cells (which
are then transduced according to a method of the invention, or are transduced
with a vector
according to the invention) is obtained from the same subject as that to which
the
transduced T-cell population is administered. Autologous transfer is
advantageous as it
avoids problems associated with immunological incompatibility and is available
to subjects
irrespective of the availability of a genetically matched donor.
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By "allogeneic cell transfer" it is to be understood that the starting
population of cells (which
are then transduced according to a method of the invention, or are transduced
with a vector
according to the invention) is obtained from a different subject as that to
which the
transduced cell population is administered. Preferably, the donor will be
genetically matched
to the subject to which the cells are administered to minimise the risk of
immunological
incompatibility. Alternatively, the donor may be mismatched and unrelated to
the patient.
Suitable doses of transduced cell populations are such as to be
therapeutically and/or
prophylactically effective. The dose to be administered may depend on the
subject and
condition to be treated, and may be readily determined by a skilled person.
The cell may be derived from a T-cell isolated from a subject. The T-cell may
be part of a
mixed cell population isolated from the subject, such as a population of
peripheral blood
lymphocytes (PBL). T-cells within the PBL population may be activated by
methods known
in the art, such as using anti-CD3 and/or anti-CD28 antibodies or cell sized
beads
conjugated with anti-CD3 and/or anti-CD28 antibodies.
The T-cell may be a CD4+ helper T cell or a CD8+ cytotoxic T cell. The cell
may be in a
mixed population of CD4+ helper T cell/CD8+ cytotoxic T-cells. Polyclonal
activation, for
example using anti-CD3 antibodies optionally in combination with anti-CD28
antibodies will
trigger the proliferation of CD4+ and CD8+ T-cells.
The cell may be isolated from the subject to which the genetically modified
cell is to be
.. adoptively transferred. In this respect, the cell may be made by isolating
a T-cell from a
subject, optionally activating the T-cell, transferring the TCR gene to the
cell ex vivo.
Subsequent immunotherapy of the subject may then be carried out by adoptive
transfer of
the TCR-transduced cells. As used herein this process refers to autologous T-
cell transfer -
i.e. the TCR-transduced cells are administered to the same subject from which
the T-cells
were originally derived.
Alternatively the T-cell may be isolated from a different subject, such that
it is allogeneic.
The T-cell may be isolated from a donor subject. For example, if the subject
is undergoing
allogeneic haematopoietic stem cell transplantation (Allo-HSCT) or solid organ
transplantation or cell transplantation or stem cell therapy, the cell may be
derived from the
donor, from which the organs, tissues or cells are derived. The donor and the
subject
undergoing treatment may be siblings.
Alternatively the cell may be, or may be derived from, a stem cell, such as a
haematopoietic
stem cell (HSC). Gene transfer into HSCs does not lead to TCR expression at
the cell
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surface as stem cells do not express CD3 molecules. However, when stem cells
differentiate into lymphoid precursors that migrate to the thymus, the
initiation of CD3
expression leads to the surface expression of the introduced TCR in
thymocytes.
An advantage of this approach is that the mature T-cells, once produced,
express only the
introduced TCR and little or no endogenous TCR chains, because the expression
of the
introduced TCR chains suppresses rearrangement of endogenous TCR gene segments
to
form functional TCR alpha and beta genes. A further benefit is that the gene-
modified stem
cells are a continuous source of mature T-cells with the desired antigen
specificity. The cell
may therefore be a gene-modified stem cell, preferably a gene-modified
hematopoeitic stem
cell, which, upon differentiation, produces a T-cell expressing a TCR of the
invention.
Other approaches known in the art may be used to reduce, limit, prevent,
silence, or
abrogate experession of endogenous genes in the cells of the invention or
cells prepared by
the methods of the invention.
As used herein the term "disrupting" refers to reducing, limiting, preventing,
silencing, or
abrogating expression of a gene. The person skilled in the art is able to use
any method
known in the art to disrupt an endogenous gene, e.g., any suitable method for
genome
editing, gene silencing, gene knock-down or gene knock-out.
For example, an endogenous gene may be disrupted with an artificial nuclease.
An artificial
nuclease is, e.g., an artificial restriction enzyme engineered to selectively
target a specific
polynucleotide sequence (e.g. encoding a gene of interest) and induce a double
strand
break in said polynucleotide sequence. Typically, the double strand break
(DSB) will be
repaired by error-prone non-homologous end joining (NHEJ) thereby resulting in
the
formation of a non-functional polynucleotide sequence, which may be unable to
express an
endogenous gene.
In some embodiments, the artificial nuclease is selected from the group
consisting of zinc
finger nucleases (ZFN), transcription activator-like effector nucleases
(TALEN) and
CRISPR/Cas (e.g. CRISPR/Cas9).
The methods of preparing a cell (e.g. a T-cell) of the invention may comprise
the step of
targeted integration of an expression cassette into an endogenous gene (e.g.
an
endogenous TCR a chain gene and/or an endogenous TCR 13 chain gene). As used
herein
the term expression cassette refers to a polynucleotide sequence (e.g. a DNA
polynucleotide
sequence) comprising one or more polynucleotide sequences encoding one or more
genes
of interest such that said genes of interest are capable of expression.
Endogenous
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sequences may facilitate expression from the expression cassete, and/or
transcription
control seuqences within the expression cassette may facilitate expression.
For example,
the expression cassette may comprise a polynucleotide sequence of the
invention, or a
polynucleotide sequence encoding a TCR of the invention, operably linked to an
expression
control sequence, e.g. a promoter or an enhancer sequence. The one or more
genes of
interest may be located between one or more sets of restriction sites.
Suitably, the
restriction sites may facilitate the integration of the expression cassette
into, e.g., a vector, a
plasmid, or genomic DNA (e.g. host cell genomic DNA).
For example, an expression cassette of the invention may be transferred from a
first
polynucleotide sequence, e.g. on a vector, to another by 'cutting', e.g.
excising, the
expression cassette using one or more suitable restriction enzymes and
'pasting', e.g.
integrating, the expression cassette into a second polynucleotide sequence.
The expression cassette may comprise a polynucleotide of the invention. The
expression
cassette may comprise a polynucleotide encoding one or more TCRs of the
invention. The
expression cassette may further comprise an antibiotic resistance gene or
other selectable
marker gene that allows cells that have successfully integrated the expression
cassette into
their DNA to be identified. The polynucleotide sequences comprised in the
expression
cassette may be operably linked to expression control sequences, e.g. a
suitable promoter
or enhancer sequence. The person skilled in the art will be able to select
suitable
expression control sequences.
The invention also contemplates a cell expressing a TCR of the invention,
which has been
engineered to disrupt one or more endogenous MHC genes. Disruption of an
endogenous
MHC gene can reduce or prevent expression of MHC on the engineered cell
surface.
Accordingly, such an engineered cell with reduced or no MHC expression will
have limited or
no capacity to present antigens on its cell surface. Such a cell is
particulary advantageous
for adoptive cell transfer since the cell will be non-alloreactive, e.g., the
cell will not present
antigens which could be recognized by the immune system of a subject receiving
the
adoptively transferred cell. As a result, the transferred cell will not be
recognized as 'non-
self' and an adverse immune reaction to the cell can be avoided. Such a cell
is termed a
.. 'universal cell' since it is suitable for adoptive transfer to a variety of
different hosts
regardless of HLA type.
Accordingly, the invention provides a method of preparing a non-alloreactive
universal T-cell,
which expresses a TCR of the invention. Further provided by the invention is a
non-
alloreactive universal T-cell, which expresses a TCR of the invention.
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The invention further contemplates cells which have been engineered to disrupt
one more
endogenous genes to modify the cell to enhance advantageous properties,
characteristics or
functions of the cell and/or reduce undesirable properties, characteristics or
functions. For
example, by disrupting an endogenous cell the persistence, expansion,
activity, resistance to
exhaustion/senescence/inhibitory signals, homing capacity, or other cell
functions may be
modified. As used in this context, the term 'modify' refers to a change in one
or more
characteristics relative to an equivalent unmodified cell, e.g. a cell in
which an endogenous
gene has not been disrupted. For example, the change may be an increase, an
enhancement or an introduction of a characteristic or function of the cell
relative to an
equivalent unmodified cell. Alternatively, the change may be a decrease,
suppression or
abrogation of a characteristic or function of the cell relative to an
equivalent unmodified cell.
The polynucleotides and vectors of the invention may be transferred into
specific T-cell
subsets, including CD4 and or CD8, naive, memory stem T cells, central memory,
effector
memory or effector cells, or in other cellular subsets such as to promote
different in vivo
length of persistence and function in the cells of the invention.
The polynucleotides and vectors of the invention may also be transferred into
T-cell subsets
such as naïve, memory stem T cells, central memory cells, effector memory
cells, effectors.
The polynucleotides and vectors of the invention may also be transferred into
T-cell subsets
with different polarizations, such as Th0/Tc0, Th1/Tc1, Th2/Tc2, Th17, Th22 or
others,
.. depending on the cytokine background most appropriate to target a
particular tumor type.
Furthermore, the polynucleotides and vectors of the invention encoding the
antigen-specific
regions of the TCRs of the present invention may be transferred in other
cellular subsets,
including gamma/delta T-cells, NK cells, NKT cells, cytokine-induced killer
(CIK) cells,
hematopoietic stem cells or other cells, in order to obtain the therapeutic
effect.
.. Further provided by the invention is a method of preparing a cell, which
comprises the step
of transducing a cell in vitro or ex vivo with a vector of the invention.
Various methods for
transduction of a cell with a vector are known in the art (see e.g. Sambrook
et al).
The invention also provides a method of producing a T-cell expressing a TCR of
the
invention by inducing the differentiation of a stem cell which comprises a
polynucleotide or a
vector of the invention.
A population of cells may be purified selectively for cells that exhibit a
specific phenotype or
characteristic, and from other cells which do not exhibit that phenotype or
characteristic, or
exhibit it to a lesser degree. For example, a population of cells that
expresses a specific
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marker (e.g. CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4) may be
purified
from a starting population of cells. Alternatively, or in addition, a
population of cells that does
not express another marker may be purified.
By "enriching" a population of cells for a certain type of cells it is to be
understood that the
concentration of that type of cells is increased within the population. The
concentration of
other types of cells may be concomitantly reduced.
Purification or enrichment may result in the population of cells being
substantially pure of
other types of cell.
Purifying or enriching for a population of cells expressing a specific marker
(e.g. CD3, CD4,
CD8, CD25, CD127, CD152, CXCR3, or CCR4) may be achieved by using an agent
that
binds to that marker, preferably substantially specifically to that marker. An
agent that binds
to a cellular marker may be an antibody, for example antibody which binds to
CD3, CD4,
CD8, CD25, CD127, CD152, CXCR3, or CCR4.
The term "antibody" refers to complete antibodies or antibody fragments
capable of binding
to a selected target, and including Fv, ScFv, F(ab') and F(ab')2, monoclonal
and polyclonal
antibodies, engineered antibodies including chimeric, CDR-grafted and
humanised
antibodies, and artificially selected antibodies produced using phage display
or alternative
techniques.
In addition, alternatives to classical antibodies may also be used in the
invention, for
example "avibodies", "avimers", "anticalins", "nanobodies" and "DARPins".
The agents that bind to specific markers may be labelled so as to be
identifiable using any of
a number of techniques known in the art. The agent may be inherently labelled,
or may be
modified by conjugating a label thereto. By "conjugating" it is to be
understood that the agent
and label are operably linked. This means that the agent and label are linked
together in a
manner which enables both to carry out their function (e.g. binding to a
marker, allowing
fluorescent identification, or allowing separation when placed in a magnetic
field)
substantially unhindered. Suitable methods of conjugation are well known in
the art and
would be readily identifiable by the skilled person.
A label may allow, for example, the labelled agent and any cell to which it is
bound to be
purified from its environment (e.g. the agent may be labelled with a magnetic
bead or an
affinity tag, such as avidin), detected or both. Detectable markers suitable
for use as a label
include fluorophores (e.g. green, cherry, cyan and orange fluorescent
proteins) and peptide
tags (e.g. His tags, Myc tags, FLAG tags and HA tags).
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A number of techniques for separating a population of cells expressing a
specific marker are
known in the art. These include magnetic bead-based separation technologies
(e.g. closed-
circuit magnetic bead-based separation), flow cytometry, fluorescence-
activated cell sorting
(FACS), affinity tag purification (e.g. using affinity columns or beads, such
as biotin columns
to separate avidin-labelled agents) and microscopy-based techniques.
It may also be possible to perform the separation using a combination of
different
techniques, such as a magnetic bead-based separation step followed by sorting
of the
resulting population of cells for one or more additional (positive or
negative) markers by flow
cytometry.
Clinical grade separation may be performed, for example, using the CliniMACS
system
(Miltenyi). This is an example of a closed-circuit magnetic bead-based
separation
technology.
It is also envisaged that dye exclusion properties (e.g. side population or
rhodamine
labelling) or enzymatic activity (e.g. ALDH activity) may be used to enrich
for HSCs.
Chimeric molecules
In another aspect, the invention provides a chimeric molecule comprising a TCR
of the
invention, a TCR encoded by a polynucleotide of the invention, or a portion
thereof,
conjugated to a non-cellular substrate. The conjugation may be covalent or non-
covalent.
The non-cellular substrate may be a nanoparticle, an exosome, or any non-
cellular substrate
known in the art.
The chimeric molecule of the invention may be soluble.
In another aspect the invention provides a chimeric molecule comprising a TCR
of the
invention, a TCR encoded by a polynucleotide of the invention, or a portion
thereof,
conjugated to a toxin or an antibody.
The toxin or antibody may be cytotoxic. The toxin may be a cytotoxic molecule
or
compound, e.g. a radioactive molecule or compound. The TCR portion of the
chimeric
molecule may confer the ability to recognize cells expressing WT1 protein or
peptides.
Thus, the chimeric molecule may specifically recognize and/or bind to WT1-
expressing
tumor cells. Accordingly, the chimeric molecules of the invention may provide
WT1-targeted
delivery of cytotoxic toxins, antibodies and/or compounds.
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WTI -related diseases
WT1 is widely expressed on a variety of hematological and solid tumors, while
showing
limited expression on various healthy tissues (e.g. gonads, uterus, kidney,
mesothelium,
progenitor cells in different tissues). The inventors have identified and
determined the amino
acid sequences of TCRs that recognise WT1 peptides. Furthermore, they have
demonstrated that T-cells expressing TCRs according to the invention target
and kill cells
which present WT1 peptide or overexpress WT1 protein.
Accordingly, the invention provides a method for treating and/or preventing a
disease
associated with expression of WT1, which comprises the step of administering a
TCR, an
isolated polynucleotide, a vector, or a cell of the invention to a subject in
need thereof. The
invention also provides a method for treating and/or preventing a disease
associated with
expression of WT1, comprises the step of administering a cell prepared by the
method of the
invention to a subject in need thereof.
Further provided by the invention is a TCR of the invention, an isolated
polynucleotide of the
invention, a vector of the invention, a cell of the invention, or a cell
prepared by the method
of the invention for use in treating and/or preventing a disease associated
with expression of
WT1.
The term 'preventing' is intended to refer to averting, delaying, impeding or
hindering the
contraction of the disease. The treatment may, for example, prevent or reduce
the likelihood
of developing or contracting a disease associated with expression of WT1.
'Treating' as used herein refers to caring for a diseased subject, in order to
ameliorate, cure
or reduce the symptoms of the disease, or in order to reduce, halt or delay
the progression
of the disease.
The subject may be a human subject. The human subject may be a child. For
example, the
child may be less than 10 years in age, less than 9 years in age, less than 8
years in age,
less than 7 years in age, less than 6 years in age, less than 5 years in age,
less than 4 years
in age, less than 3 years in age, or less than 2 years in age. The human
subject may be an
infant.
The subject may have been previously determined to be in need of a TCR, an
isolated
polynucleotide, a vector, or a cell of the invention, or a cell prepared by
the method of the
invention on the basis of expression of WT1. For example, the subject may have
a cell
population that exhibits increased expression of WT1 relative to a healthy
control cell
population. A variety of techniques known in the art may be used to determine
WT1
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expression - e.g. quantitative RT-PCR can be used to determine the amount of
WT1 RNA
transcript, which is indicative of WT1 protein expression. The person skilled
in the art will
also appreciate that WT1 protein expression may be determined by performing
western blots
using commercially available antibodies specific for WT1.
The subject may also have been previously identified as having an alteration
(e.g. mutation
or deletion) in a WT1 gene. Such an alteration may be hereditary. Thus, the
disease
associated with expression of WT1 may be a hereditary disease. Examples of
hereditary
disases associated with expression of WT1 include but are not limited to WAGR
(Wilms
tumor-Aniridia-Genitourinary malformation-Retardation) syndrome, Denys-Drash
syndrome
(DDS), Frasier syndrome (FS), genitourinary anomalies (abnormalities of the
reproductive
and urinary systems) syndrome.
Subjects with hereditary disases associated with expression of WT1 may be at
higher risk of
developing a proliferative disorder (e.g. a cancer).
The disease associated with expression of WT1 may be a proliferative disorder.
The proliferative disorder may be a hematological malignancy or a solid tumor.
The
hematological malignancy may be selected from the group consisting of acute
myeloid
leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia,
myelodisplastic
syndromes, lymphoma, multiple myeloma, non Hodgkin lymphoma, and Hodgkin
lymphoma.
The solid tumor may be selected from the group consisting of lung cancer,
breast cancer,
oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma,
pancreatic cancer,
ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma,
osteosarcoma,
thyroid cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver
cancer,
melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial
cancer, biliary
cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.
The disease associated with expression of WT1 may be selected from a group
consisting of
acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic
leukemia,
myelodisplastic syndromes, lymphoma, multiple myeloma, non Hodgkin lymphoma,
and
Hodgkin lymphoma, lung cancer, breast cancer, oesophageal cancer, gastric
cancer, colon
cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck
cancers,
synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial
cancer,
neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal
cancer,
soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma,
mesothelioma, cervical
cancer, and colorectal cancer.
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Pharmaceutical composition
The TCRs of the invention, the polynucleotides of the invention, the vectors
of the invention,
the cells of the invention, the cells prepared by the methods of the
invention, the chimeric
molecules of the invention, and the mixed cell population of the invention may
be formulated
for administration to subjects with a pharmaceutically acceptable carrier,
diluent or excipient.
Suitable carriers and diluents include isotonic saline solutions, for example
phosphate-
buffered saline, and potentially contain human serum albumin.
Handling of the cell therapy products is preferably performed in compliance
with FACT-
JACIE International Standards for cellular therapy.
Method of treatment
In another aspect, the invention provides a method for treating and/or
preventing a disease
associated with expression of WT1, which comprises the step of administering a
TCR of the
invention, an isolated polynucleotide of the invention, a vector of the
invention, a cell of the
invention, a cell prepared by a method of the invention, a chimeric molecule
of the invention,
or a mixed cell population of the invention to a subject in need thereof.
The subject may be a human subject. The subject may be a non-human animal
subject.
The subject may have a disease associated with expression of WT1. The subject
may be at
risk of developing a dieases associated with expression of WT1. The subject
may have been
previously determined to be at risk of developing a disease associated with
expression of
WT1. The subject may have an increased risk of developing a disease associated
with
WT1.
The increased risk may have been determined by genetic screening and/or by
reviewing the
subject's family history. The subject may express genetic markers indicative
of increased
risk of developing a disease associated with expression of WT1.
Suitably, a person skilled in the art will be aware of genetic risk factors
(e.g. genetic markers)
associated with increased risk of developing a disease associated with WT1.
The skilled
person may be able to use any suitable method or technique known in the art to
determine
whether the subject has an increased risk of developing a disease associated
with
expression of WT1.
The subject may have previously received treatment for a disease associated
with
expression of WT1. The subject may be in remission. The subject may be
resistant to
chemotherapy. The subject may be resistant to an anti-WT1 therapy.
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In one embodiment, the method for treating and/or preventing a disease
associated with
expression of WT1 comprises the step of administering a chemotherapy to the
subject. The
chemotherapy may be administered to the subject simultaneously, sequentially
or separately
with the TCR of the invention, the isolated polynucleotide of the invention,
the vector of the
invention, the cell according of the invention, the cell prepared by the
method of the
invention, or the chimeric molecule of the invention.
In another aspect, the invention provides a method of treating and/or
preventing a disease
associated with expression of WT1, which comprises the step of administering a
mixed cell
population, wherein the mixed cell population comprises a plurality of cell
populations each
expressing a different TCR of the invention.
In another aspect, the invention provides a mixed cell population comprising a
plurality of cell
populations each expressing a different TCR of the invention.
In another aspect, the invention provides a method for preparing a mixed cell
population
comprising a plurality of cell populations each expressing a different TCR of
the invention,
wherein the method comprises the step of transducing a cell in vitro or ex
vivo with a vector
of the invention.
In another aspect, the invention provides a mixed cell population for use in
treating and/or
preventing a disease associated with expression of WT1, wherein the mixed cell
population
comprises a plurality of cell populations each expressing a different TCR of
the invention.
For example, the mixed cell population may comprise a first cell population
expressing a first
TCR of the invention and a second cell population expressing a second TCR of
the
invention. For example, the mixed cell population may comprise a first cell
population
expressing a first TCR of the invention, a second cell population expressing a
second TCR
of the invention, and a third cell population expressing a third TCR of the
invention, and so
on.
Each cell population of the mixed cell population may, for example, express a
single TCR of
the invention only. The endogenous TCR genes of the cell populations in the
mixed cell
population may be disrupted or deleted. Expression of endogenous TCR genes of
the cells
in the mixed cell population may be disrupted, e.g. by gene editing with an
artificial nuclease.
In another aspect, the invention provides use of TCR of the invention, an
isolated
polynucleotide of the invention, a vector of the invention, a cell of the
invention, a cell
prepared by a method of the invention, a chimeric molecule of the invention,
or a mixed cell
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population of the invention, for the manufacture of a medicament for the
treatment of a
disease associated with expression of WT1.
Both human and veterinary treatments are within the scope of the invention.
The practice of the invention will employ, unless otherwise indicated,
conventional
techniques of cell biology, molecular biology, histology, immunology,
oncology, which are
within the capabilities of a person of ordinary skill in the art. Such
techniques are explained
in the literature.
See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)
Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel,
F.M. et al.
(1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9,
13 and 16,
John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and
Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee,
J.O'D.
(1990) In Situ Hybridization: Principles and Practice, Oxford University
Press; Gait, M.J.
(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and LiIley,
D.M. and
Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis
and
Physical Analysis of DNA, Academic Press. Each of these general texts is
herein
incorporated by reference.
Various preferred features and embodiments of the invention will now be
described by way
of non-limiting examples.
EXAMPLES
EXAMPLE 1
Materials and methods
Peptides
The WT1 protein sequence previously published by Gessler et al. (Doubrovina,
E. et al.
Blood 120: 1633-1646 (2012)) was adopted to design the peptides used for the
stimulation
and isolation of WT1-specific T cells. This sequence contains 575 amino acids
and includes
the first 126 amino acids in the N-terminus missing in the (exon 5+, KTS+)
isoform of WT1. It
is composed of 141 pentadecapeptides spanning the whole sequence of the WT1
protein,
each overlapping the next one by 11 amino acids. Starting from the original
pool described in
Doubrovina et al., in order to increase the probability to enrich for WT1-
specific T cells
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restricted to peptides processed and presented by different HLA alleles (and
in particular by
the HLA-A*02:01 restriction element), we used 3 different protocols.
1. Stimulation with WT1 pool-137:
For Healthy Donor 12 (HD12), peripheral blood mononuclear cells (PBMCs) were
stimulated with a WT1 pool of 137 pentadecapeptides (indicated as WT1 p001-
137)
obtained by excluding peptides 40, 41, 63, 64 in order to avoid the isolation
of T cells
specific for the WT1 37-45 epitope (VLDFAPPGA (SEQ ID NO: 72), an
immunodominant peptide restricted to the HLA-A*02:01 allele) and the WT1 126-
134
epitope (RMFPNAPYL (SEQ ID NO: 71), an immunogenic peptide which has been
described to be processed by the immunoproteasome (Jaigirdar, A. et al. J
lmmunother. 39(3):105-16 (2016) and presented by the HLA-A*02:01 allele).
2. Stimulation with WT1-HLA-A*02:01 pool:
For HD13, HD14, HD15, PBMCs were stimulated with a pool composed of defined
peptides already known to be possibly restricted to the HLA-A*02:01 allele
(Doubrovina, E. et al. Blood 120: 1633-1646 (2012)). Selected peptides
indicated in
Table 3 were pooled at a concentration of 13.6 pg/ml per peptide. These
peptides
are labelled according to the nomenclature already used for the WT1 pool (141
peptides) described above (indicated as WT1-HLA-A*02:01 pool). We did not
include
in the new pool peptides VLDFAPPGA (SEQ ID NO: 72) (P40-41) and RMFPNAPYL
(SEQ ID NO: 71) (P63-64).
3. Stimulation with a single peptide:
PBMCs of HD15 were also stimulated with a single peptide (P91) chosen for its
HLA-
restriction (possibly HLA-A*02:01), its natural processing and its expression
on
primary leukemic blasts (as reported in Doubrovina et al.).
Peptides were synthesised by PRIMM to specifications of validated sequence,
70% purity,
sterility and absence of endotoxin. These peptides were mixed in equal amounts
in the WT1
pool composed of 137 peptides (WT1 p001-137) at a concentration of 1 pg/ml per
peptide.
Additionally, 24 subpools were generated, each containing up to 12 peptides
(4.17 pg/ml per
peptide) according to a specific mapping matrix in order to have each peptide
included in
only two overlapping subpools as shown in Table 4.
Table 3. Peptides included in the WT1 HLA-A*02:01 pool.
Peptide number Peptide sequence SEQ ID NO
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P4 PTACPLPHFPPSLPP SEQ ID NO: 80
P7 LPPTHSPTHPPRAGT SEQ ID NO: 81
P13 LLAAILDFLLLQDPA SEQ ID NO: 82
P20 RSGPGCLQQPEQQGV SEQ ID NO: 83
P25 IWAKLGAAEASAERL SEQ ID NO: 84
P33 SDVRDLNALLPAVPS SEQ ID NO: 85
P37 GGGGGCALPVSGAAQ SEQ ID NO: 86
P91 CMTWNQMNLGATLKG SEQ ID NO: 87
P92 NQMNLGATLKGVAAG SEQ ID NO: 88
P129 TCQRKFSRSDHLKTH SEQ ID NO: 89
P131 SDHLKTHTRTHTGKT SEQ ID NO: 90
Table 4. Mapping grid strategy.
SP1 SP2 SP3 SP4 SP5 SP6 SP7 SP8 SP9 SP10
SP11 SP12
SP13 1 2 3 4 5 6 7 8 9 10 11 12
SP14 13 14 15 16 17 18 19 20 21 22 23 24
SP15 25 26 27 28 29 30 31 32 33 34 35 36
SP16 37 38 39 40 41 42 43 44 45 46 47 48
SP17 49 50 51 52 53 54 55 56 57 58 59 60
SP18 61 62 63 64 65 66 67 68 69 70 71 72
SP19 73 74 75 76 77 78 79 80 81 82 83 84
SP20 85 86 87 88 89 90 91 92 93 94 95 96
SP21 97 98 99 100 101 102 103 104 105 106
107 108
SP22 109 110 111 112 113 114 115 116 117 118
119 120
SP23 121 122 123 124 125 126 127 128 129 130
131 132
SP24 133 134 135 136 137 138 139 140 141
Isolation of peripheral blood mononuclear cells
Peripheral blood was obtained from 4 healthy donors (HDs) at San Raffaele
Hospital (OSR)
upon informed consent. Peripheral blood mononuclear cells (PBMCs) were
isolated using
Ficoll-Hypaque density gradient centrifugation.
Immortalised B cells
Autologous B cells were isolated from PBMCs of healthy donors using the CD19
Microbeads
(Miltenyi Biotec). Cells were transduced with a lentiviral vector harboring
the BCL-6/BCL-XL
.. transgene (Kwakkenbos, M. J. et al. Nat. Med. Jan;16(1):123-(2010)) and the
H/F
pseudotype (Levy, C. et al. Molecular Therapy20 9, 1699-1712, (2012) and
cultured in
lscove's Modified Dulbecco's Medium (IMDM) (Euroclone/Lonza) supplemented with
10%
fetal bovine serum (FBS; Carlo Erba), 1% penicillin-streptomycin
(Euroclone/Lonza), 2 mM
glutamine and 50 ng/ml of IL21 (Miltenyi Biotec). B-cells were re-stimulated
every 5 days by
co-culture with irradiated (80 Gy) mouse L-cell fibroblasts expressing CD4OL
(3T3-CD4OL) at
a B-ce11:3T3-CD4OL ratio of 10:1.
Cell lines
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T2 and EBV-BLCLs cell lines were cultured in IMDM (Euroclone/Lonza) both
supplemented
with 1% penicillin-streptomycin, 2mM glutamine and 10% FBS.
Leukemic cells
Primary AML cells were obtained from the OSR Leukemia biobank and selected
according
to the expression of WT1 (determined by quantitative PCR) and of the HLA
typing. In co-
culture experiments, leukemic blasts were kept in X-VIVO 15 (Euroclone/Lonza)
medium
supplemented with 5% HS, 1% penicillin-streptomycin, 2 mM glutamine, IL3 and G-
CSF
(Peprotech; both 20 ng/ml).
HLA typing
Healthy donor samples, Epstein-Barr virus (EBV)-B lymphoblastoid cell lines
(BLCLs) and
primary leukemic cells were typed for HLA-A, HLA-B, HLA-C alleles at high
resolution at the
HLA laboratory of OSR.
Flow cytometty
FITC-, PE-, PerCP-, APC-, PE-Cy7, APC Cy7-, Pacific Blue and Brillant
Violet¨conjugated
antibodies directed to human CD3, CD4, CD8, CD107a, interferon (IFN)y, Tumor
necrosis
factor (TNF)a, CD33, CD117, CD34, CD14, anti-active Caspase 3, and HLA-A2 were
used.
Cells were incubated with antibodies for 15 minutes at 4 C and washed with
phosphate-
buffered saline (PBS) containing 1% FBS. For Caspase 3 staining, cells were
incubated for
one hour at 4 C. Zombie Aqua Fixable Viability kit (Biolegend) was used to
stain dead cells
according to the manufacturer's instructions. Flow cytometry data were
acquired using one
of the following cell analysers: BD Canto ll flow cytometer, BD LSRFortessa,
Cytoflex S
(Beckman Coulter). Data were analysed by Flow Jo software (Tree star Inc). For
intracellular
evaluation of cytokine secretion and expression of degranulation markers, the
Fix/Perm
buffer set (Biolegend) was used according to the manufacturer's instructions.
Stimulation, isolation and expansion of WT1-specific T-cells
Freshly isolated PBMCs were resuspended in X-VIVO 15 (Euroclone/Lonza)
supplemented
with 5% human AB serum, 1% penicillin-streptomycin, 2 mM glutamine and 1 pg/ml
CD28
monoclonal antibody (BD Biosciences), seeded at a density of 107 cells/ml and
stimulated
with: 1) WT1 pool-137 for HD12, 2) WT1-HLA-A*02:01 pool for HD13-HD14-HD15, 3)
single
peptide (P91) for HD15.
For experiments performed with 1) and 2), antigen-specific T-cells were
isolated after 26-30
hours by CD137 expression. More specifically, cells were stained with the PE-
conjugated
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CD137 antibody and sorted using anti-PE microbeads (Miltenyi Biotec). The
CD137- fraction
was depleted of the CD3 cells using CD3-Microbeads (Miltenyi Biotec),
irradiated 30 Gy and
used as peptide-loaded antigen presenting cells (APCs) in a co-culture with
the CD137+
fraction at a ratio of 100:1 when possible or at least 20:1 and a final
density of 5x106
cells/ml. X-VIVO 15 supplemented with 5% human AB serum, 1% penicillin-
streptomycin, 2
mM glutamine, 5 ng/ml IL7, 5 ng/ml IL15 and 10 ng/ml IL21 was used as medium.
Media,
including cytokines, was replaced every 2-3 days.
For the experiment performed with 3), antigen-specific T cells were stimulated
with P91
epitope in RPM! (Euroclone/Lonza) supplemented with 5% human AB serum. After 6
hours,
cells were harvested, washed with PBS, labelled with the IFNy-catch reagent
and incubated
for 45 minutes at 37 C. Afterwards, cells were stained with a PE-labelled
antibody to IFNy,
enriched by using anti-PE microbeads and separated using the MACS system
(Miltenyi
Biotec). IFNy-enriched T cells were co-cultured with IFNy - CD3- fraction
irradiated with 30
Gy at a ratio of 100:1 and seeded at a density of 5x106 cells/ml. X-VIVO 15
supplemented
with 5% human AB serum, 1% penicillin-streptomycin, 2 mM glutamine, 5 ng/ml
IL7, 5 ng/ml
IL15 and 10 ng/ml IL21 was used as medium. Media, including cytokines, was
replaced
every 2-3 days.
After ¨20 days T cells were pelleted and used for TCR sequencing analysis.
Re-stimulation of expanded antigen-specific T-cells
Cells originally stimulated using either protocol 1) or 2) (as described
above) were re-
stimulated every 7-14 days with WT1-pulsed autologous APCs (PBMC CD3-depleted
cells).
In the initial re-stimulations, cells were washed 2 days before and plated in
cytokine-free
medium. APCs were irradiated with 30 Gy, pulsed with the peptide pool
overnight in X-VIVO
15 supplemented with 5% AB serum or at least 3 hours on a rotator in IMDM
without serum.
Pulsed APCs were co-cultured with effector cells in X-VIVO 15 supplemented
with 5%
human AB serum, 1% penicillin-streptomycin, 2 mM glutamine, 1 pg/ml CD28
monoclonal
antibody and IL7 (5 ng/ml), IL15 (5 ng/ml), IL21 (10 ng/ml).
Assessment of T cell response
The percentage of T-cells responding to the WT1 pool-137 or to the WT1-
HLA*A02:01 pool
was measured by performing a 6 hour co-culture of the effector cells with
autologous APCs
(ratio of at least 1:1) pulsed with the desired antigen (WT1 pool-137 or WT1-
HLA*02:01 pool,
WT1 subpools or unrelated peptide pool as control). Co-cultures were seeded in
X-VIVO 15
supplemented with 5% human AB serum, 1% penicillin-streptomycin, 2 mM
glutamine and
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supplemented with the CD28 monoclonal antibody (1 pg/ml), Golgi Stop Protein
transport
inhibitor (BD Biosciences; 1 pg/ml) and CD107a-FITC antibody (BD Biosciences;
4 p1/well)
for assessment of degranulation. Cells were then fixed, permeabilised and
stained
intracellularly to determine the percentage of CD3+CD8+ or CD3+CD4+ cells
secreting IFNy
and expressing CD107a.
Mapping of immunogenic peptides
WT1-specific T-cells of HD12 enriched using the WT1 pool-137 were seeded in
different
wells and co-cultured with autologous APCs loaded with one of each of the WT1
subpools.
WT1-specific T-cells of HD13 and HD14, enriched using the WT1 HLA-A*02:01 pool
were
seeded in different wells and co-cultured with autologous APCs loaded with the
individual
peptides included in the WT1-HLA*A02:01 pool. For HD15, mapping of immunogenic
peptides was not performed due to a reduced cellularity.
Each co-culture was seeded at an effector to target ratio of at least 1:1. T-
cell responses to
each subpool or peptide were measured as previously described by FACS
analysis. For
HD12, deconvolution of the mapping grid was essential to determine which
shared peptide
was eliciting a T cell response. Once determined the immunogenic epitopes, T-
cells of
HD12, HD13, HD14 were further stimulated with APCs loaded with the individual
peptides.
Evaluation of T cell ability to recognise WT1-expressing cells
WT1-specificity and HLA-restricted ability of T-cells to recognise target
cells was measured
with different experimental procedures. For HD13 and HD14, the percentage of
living target
cells expressing the Caspase 3 were determined. Primary leukemic blasts and T
cells were
incubated at an effector to target (E:T) ratio of 10:1, 4:1, 1:1, 1:4 and 1:10
for 6 hours. As a
negative control, target cells were cultured with unrelated T lymphocytes.
Cells were fixed,
permeabilised using the Fix/Perm buffer set (Biolegend) and stained with anti-
active
Caspase-3¨antibody conjugated to Pacific Blue (Biolegend). Dead cells were
visualised
upon staining with Zombie Aqua Fixable Viability kit (Biolegend).
For the remaining donors, due to a reduced fitness of the expanded WT1-
specific T cells, it
was not possible to perform these functional assays.
Enrichment of IFNy-secreting cells
In order to enrich T cells specific for WT1 expanded from HD13, we performed
the IFNy
capture assay (Miltenyi Biotec). Briefly, T cells were stimulated with the
immunogenic
recognised epitope for 6 h. Cells were harvested, washed with PBS and labelled
with the
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IFNy-catch reagent. After 45 minutes incubation at 37 C, cells were stained
with a PE-
labelled antibody to the IFNy. IFNy-secreting cells were afterwards enriched
by using anti-
PE microbeads and separated using the MACS system (Miltenyi Biotec). IFNy-
enriched T
cells were expanded using the protocol described in the following paragraph.
Expansion of WT1-specific T cells
Upon several restimulations with autologous APCs, to further expand WT1-
specific T cells
from HD12, HD13, HD14, different protocols were used.
For HD12 and HD13, a rapid expansion protocol (REP) was used as previously
described
(Riddell, S. R. et al. Science 80 (1992); ME, D., LT, N., Westwood, J., JR, W.
& SA, R.
Cancer J. (2000)).
For HD14, WT1-expanded T cells were stimulated with allogeneic irradiated (30
Gy) feeder
cells derived from 3 different donors (2 of them harbouring the HLA-A*0201
allele) as well as
T2 irradiated (100 Gy) cells both pulsed with the P13 peptide
(effector:T2:feeder ratio=1:5:1).
Assessment of T cell clonality
In order to determine the clonality of the expanded WT1-specific T cells, the
10 Test Beta
Mark TCR V beta repertoire kit (Beckman Coulter) was used according to the
manufacturer's
recommendations.
TCR repertoire sequencing
WT1-specific T cells were collected at different time points over the co-
culture time frame
and RNA was extracted by using the Arcturus Pico Pure RNA extraction kit (Life
Technology). Complementarity determining region (CDR) 3 sequences of the WT1-
specific T
cells were amplified by using a modified RACE approach (Ruggiero,E. et al.
Nat. Commun.
6,8081 (2015)). Samples were sequenced by using an Illumine MiSeq sequencer
and CDR3
clonotypes identified using the MiXCR software (Bolotin, DA et al. Nature
Methods 12, 380-
381 (2015)).
Lentiviral vectors
TCR a and 13 chain genes isolated from HD12, HD13, HD14 and HD15 were codon-
optimised, cysteine-modified (Kuball, J. et al. (2007) Blood 109: 2331-8) and
cloned in a
lentiviral vector (LV) under a bidirectional promoter (European Patent No.
1616012). For
WT1-specific T cells originating from HD14 the MiXCR analysis revealed the
occurrence of 3
possible TRAV genes in the generation of the same CDR3 region recognising
peptide 13.
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Thus, we ordered 3 different TCR constructs harbouring one of the following
genes:
TRAV12-3*01, TRAV12-2*01, TRAV12-2*02. For TCRs harbouring TRAV12-2*01 and
TRAV12-2*02 genes, we tested: 1) codon optimised, cysteine-modified forms and
2) codon
optimised, cysteine-modified forms further mutagenised in order to remove one
N-
glycosylation site in the TCR alpha constant domain (Kuball, J et al. (2009) J
Exp Med 206:
463-75). In particular, we substituted the amino acid N at position 36 in a N-
X-S/T motif with
the amino acid Q.
HD14-derived TCRs were named as follows:
TRAV 12-3*01 - cysteine modified, codon optimized
TRAV12-2*01 WT - cysteine modified, codon optimized
TRAV12-2*01 mut - cysteine modified, codon optimized, mutagenized to remove a
N
glycosylation site
TRAV12-2*02 WT - cysteine modified, codon optimized
TRAV12-2*02 mut - cysteine modified, codon optimized, mutagenized to remove a
N
glycosylation site
For each TCR, the alpha chain was cloned in antisense orientation under the
minimal
human CMV promoter and the beta chain in sense orientation under the PGK
promoter. LVs
were packaged by an integrase-competent third-generation construct and
pseudotyped by
the vescicular stomatitis virus (VSV) envelope.
Vector transductions
For transduction with HD13- and HD14-TCR lentiviral vectors, T lymphocytes
isolated from
healthy individuals were activated and sorted using magnetic beads conjugated
to antibodies
to CD3 and CD28 (ClinExVivo CD3/CD28; lnvitrogen), following the
manufacturer's
instructions. Cells were seeded at a concentration of 1-2x106 cells/ml and
cultured in IMDM
supplemented with 1% penicillin, 1% streptomycin, 10% FBS and 5 ng/ml of each
IL-7 and
IL-15. For transduction, T lymphocytes were plated at 2.5 x 106 cells/ml and
infected with the
LV for 24 h. Afterwards, T cells were cultured at 106 cells/ml and expanded.
Transduction
efficiency was determined by measuring the percentage of the CD3+ T cells
expressing the
specific V13 (HD13: no antibody available for the V13; HD14: V[312).
TCR editing of T lymphocytes
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PBMCs from HDs were activated and sorted using magnetic beads conjugated to
antibodies
to CD3 and CD28 (ClinExVivo CD3/CD28; lnvitrogen) and seeded at a
concentration of 1-
2x106 cells/ml in X-VIVO 15 supplemented with 1% penicillin, 1% streptomycin,
5% FBS and
ng/ml of each IL-7 and IL-15. After 2 days, T cells were electroporated with
RNP
5 complexes (originated from the combination of TRAC or TRBC guides and
Cas9 protein)
simultaneously. Edited T lymphocytes were transduced at day 3 with a LV
encoding for the
HD12-, HD13- and HD14-derived TCRs. After 6 days, beads were detached and
cells were
seeded at a concentration of 1x106 cells/ml. After 14 days, transduction
efficiency was
determined by measuring the percentage of CD3+ T cells expressing the specific
V13
(HD12:V[322; HD13: no antibody available for the V13; HD14: V[312). HD12-
edited T cells
were stained with MIX G (containing anti-V1322 antibody conjugated to FITC
fluorochrome-
10 Test Beta Mark kit, Beckman Coulter) and sorted using anti-FITC Microbeads
(Miltenyi
Biotec) following the manufacturer's instructions.
Functional assays with engineered T lymphocytes
The ability of HD12, HD13 and HD14-engineered T cells (either by TCR gene
transfer or
TCR gene editing) to recognise target cells was measured upon co-culture with:
(a) for HD13
and HD14 TCRs, T2 cells either pulsed with a peptide pool (WT1 pool or an
unrelated one)
or with subpools (1 and 14, both containing peptide 13, or with an unrelated
one) at an (E)
effector:target (T) ratio of 1:1; (b) for HD12 TCR, an EBV cell line
harbouring the HLA-
.. C*07:02 allele pulsed with peptide 103 or with an unrelated peptide as
control; (c) for HD14
TCR, primary AML blasts selected according to the expression of the HLA-A*0201
allele and
of the WT1 antigen (at different E:T ratios, i.e. 50:1; 5:1). After 6 hour co-
culture, the
percentage of responding cells was determined by evaluating CD107a expression
and/or
IFNy secretion on CD8+ T lymphocytes by cytofluorimetric analysis for assays
involving T2
cells or EBV cell lines and active Cas3 expression on living target cells for
assays involving
primary AML blasts.
Results
Generation of functional WT1-CTLs from healthy donors.
We stimulated PBMCs from HD12 using a pool of 137 pentadecapeptides (WT1 p001-
137),
which differs from the original pool described by Doubrovina et al. as we
excluded peptides
40, 41, 63, 64. These peptides were excluded in order to avoid the isolation
of T cells
specific for the WT1 37-45 epitope (VLDFAPPGA, SEQ ID NO: 72) and the WT1 126-
134
epitope (RMFPNAPYL, SEQ ID NO: 71).
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Furthermore, we stimulated PBMCs from additional 3 donors (HD13-HD15) with the
WT1-
HLA-A*02:01 pool. After 26-30 hours, CD137+ T cells were sorted and co-
cultured with the
CD137- population, further depleted of the CD3 fraction, and irradiated at 30
Gy.
Cells were repetitively stimulated with APCs represented by CD3- cells loaded
with the
peptide pool. Expansion of WT1-specific T cells was evaluated over time by
cytofluorimetric
analysis to assess cytokine release (IFNy, IL-2, TNF-a) and expression of
degranulation
marker (CD107a). As a negative control, cells were stimulated with a peptide
pool originated
from an unrelated antigen. Overall, we observed expansion of tumor-specific T
lymphocytes
in the CD8 fraction upon at least three stimulations with WT1 pool (Figure 1,
a,b,c,d).
For HD15, in a separate experiment, we stimulated PBMCs with a single peptide
(P91) and
enriched WT1-specific T cells by using the IFNy catch assay. Upon ¨20 days of
culture, T
cells were used for TCR sequencing analysis.
Mapping of WT1 epitopes eliciting a T cell response.
In order to identify which pentadecapeptide of the WT1 pool-137 elicited an
immune
.. response in HD12, we used a mapping grid strategy as previously described
by Doubrovina
et al. Briefly, WT1 overlapping pentadecapeptides were subdivided into 24
subpools (SPs)
containing up to 12 peptides in which each peptide of the 141 peptides
described in
Doubrovina et al. was uniquely contained within two intersecting SPs. Enriched
WT1-specific
T cells were co-cultured for 6 hours with irradiated APCs (autologous
immortalised B cells)
pulsed with the 24 SPs and we measured the percentage of IFNy secretion and
CD107a
expression by flow cytometry. This strategy enables the detection of the
immunogenic
peptide by the deconvolution of the mapping grid. For HD13 and HD14, we
stimulated
autologous APCs with each of the individual peptides included in the WT1 HLA-
A*02:01 pool
and used them as target cells in a 6-hour co-culture experiment with WT1-
specific T cells.
For HD15, WT1-enriched T cells originated upon stimulation of the PBMCs with
the WT1
HLA-A*02:01 pool, we did not perform the mapping of the immunogenic peptides
due to the
reduced cellularity.
We observed a substantial secretion of IFNy and expression of CD107a after
stimulation of
T cells with subpools SP7 and SP21 (Figure 2a, b). For HD13 and HD14 (Figure
2c),
stimulated with the WT1 pool HLA-A*02:01, there was a robust immune response
towards
autologous APCs pulsed with the P13 peptide (Figure 2d, e for HD13 and HD14,
respectively).
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Once identified for HD12 the SP recognised by WT1-specific T cells, T
lymphocytes were re-
stimulated using CD3-depleted PBMCs pulsed with the specific peptide
identified upon
deconvolution of the mapping grid (Figure 3a; highlighted is the immunogenic
peptide
identified).
In a stepwise approach, to validate the immunogenic pentadecapeptide, we
tested T cells in
a 6 hour co-culture with autologous irradiated immortalised B cells loaded
with the 15mer
eliciting the immune response and with at least one unrelated 15mer. Increased
expression
of CD107a and IFNy secretion was observed for peptide 103 (Figure 3b). The
identified
immunogenic pentadecapeptide was further used to re-stimulate T cells in order
to provide
an enrichment of the epitope-specific population. For HD13 and HD14, T cells
were
restimulated with autologous APCs stimulated with the recognised peptide
(P13).
In silico prediction of peptide-MHC binding
In order to predict for each HD characterised by the expansion of CD8-specific
T cells the
exact binding nonamers and their HLA-restrictions, we used the NetMHCpan 4.0
server
(Jurtz V. et al. (2017) The Journal of Immunology). Binding prediction was
performed only for
peptides presented by HLA class I molecules, which have a strong preference
for peptides
of 9 amino acids. A defined peptide will be identified as a strong binder if
the % Rank is
below the 0.5% and as weak binder if the % Rank is between 0.5% and 2%.
In order to determine HLA-alleles harboured from HD12-HD15, DNA of each
individual was
HLA-typed (for HLA-A, HLA-B, HLA-C alleles) at high resolution in the HLA and
Chimerism
Laboratory of Ospedale San Raffaele (Figure 4a).
For HD12, 2 strong binders were identified: peptide YRIHTHGVF (SEQ ID NO: 73)
either on
the HLA-B*38:01 allele or on the HLA-C*07:02 allele (stronger binding) (Figure
4b).
For HD13 and HD14, peptide LLAAILDFL (SEQ ID NO: 74) was identified as a
strong binder
in combination with the HLA-A*02:01 allele (Figure 4c, d); in addition for
HD14, peptide
AAILDFLLL (SEQ ID NO: 75) was evidenced as a strong binder when presented by
HLA-
C*03:03 allele (Figure 4d).
For HD15, no strong binders were predicted.
Identified WT1 peptides represent immunogenic peptides presented by different
HLA alleles
In order to determine the HLA restriction of the antigen-specific T cells
identified for each HD
analysed, T lymphocytes were co-cultured with target cells expressing (or not)
specific HLA
class I alleles harboured by the HD.
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For HD12, we used as a target a panel of EBV-BLCLs harbouring a single HLA
allele in
common with the HD, pulsed either with the relevant peptide or with an
unrelated one.
Results showed an increase in the number of cells expressing CD107a and
secreting IFNy
upon co-culture with each EBV-BLCLs harboring the HLA-C*07:02 allele and
pulsed with the
WT1 P103 peptide (Figure 5a).
For HD13 and HD14, stimulated with a pool of peptides previously reported to
be able to
elicit an immune response when presented by the HLA-A*02:01 allele, we
directly performed
a functional validation by co-culturing with T2 cells pulsed either with the
specific
immunogenic epitope (P13) or with an unrelated one. Flow cytometry results
showed a great
increase in the percentage of cells expressing the CD107a marker and secreting
IFNy upon
co-culture of T lymphocytes with T2 cells pulsed with P13 (Figure 5, b, c).
Assessment of peptide processing by FA CS.
To determine the ability of WT1-expanded T cells isolated from HD13 (Figure
6a) and HD14
(Figure 6b) to recognise a naturally processed peptide and kill target cells,
we evaluated the
expression of active Caspase 3 in living primary blasts upon 6 hour co-culture
with T
lymphocytes. As target cells we used primary blasts from 3 AML patients
selected according
to the high expression of the WT1 antigen and to the HLA typing (HLA-A*02:01).
As a
control, we included co-cultures of unrelated effector cells with the same
leukemic blasts
used for HD13 and HD14. Results showed the ability of both HD13 as well as
HD14 T cells
to recognise primary leukemic blasts, with HD14 showing a greater elimination
of AML blasts
at the different effector to target ratios used.
For HD12, due to the low cellularity of the cell population we did not perform
any test to
validate the natural processing of the recognised peptide.
Overall, the ability of the WT1-specific T cells originated from HD13 and HD14
to recognise
WT1-expressing target cells (leukemic blasts) indicated not only the natural
processing of
the recognised peptide but also its immunogenicity.
Immunoprofiling of WT1-specific T cells.
To characterise the newly identify WT1-specific TCRs, we performed both flow
cytometry of
the TCR V13 families and TCR sequencing. FACS results indicated the prevalence
of a
specific V13 for HD12 and 14; for HD13 an exhaustive determination of the
predominant V13
was not possible, as the 10 Test Beta Mark TCR V beta repertoire guarantees a
coverage of
75% of the complete repertoire of V beta (Figure 7). For HD15 WT1-specific T
cells, the flow
cytometry assessment of the expressed V13 families was not performed due to
the low
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cellularity and the reduced cell fitness. TCRa8 sequencing of WT1-specific T
cells
highlighted the increasing predominance of one CDR3 clonotype over time for
both TCR
chains in HD12, HD13, HD14 (Figure 8a-c). For HD15, we observed a clear
expansion of
specific TCR chains both upon stimulations with the WT1 HLA-A*02:01 pool and
upon
stimulation with the individual peptide (P91) followed by IFNy enrichment
(Figure 8d).
Functional validation of the newly cloned TCRs
TCR a and 13 sequences isolated from HD12, HD13, HD14 and HD15 and recognising
WT1
epitopes restricted to HLA class I alleles were further modified in order to
increase their
surface expression and reduce mispairing with endogenous TCR chains. For HD14
TCRs
we further mutagenised the receptors in order to increase their functional
avidity as
described in Kuball, J et al. (2009) J Exp Med 206: 463-75. TCR genes obtained
from HD12,
HD13 and HD14 (all different forms generated as described in Materials and
Methods of
Example 1) were cloned into bidirectional lentiviral vectors to promote robust
and coordinate
expression of both TCR chains in transduced lymphocytes. Viral production for
lentiviral
vectors encoding cloned TCRs was performed.
T cells from healthy individuals were transduced with lentiviral vectors
previously generated
from HD12, HD13 and HD14.
For HD12 TCR, activated T cells originating from 3 different healthy donors
were edited as
described in the Materials and Methods section (Example 1). T cells were
transduced with
the LV encoding the specific TCR a and 13 chain genes upon disruption of the
endogenous
TCR repertoire. After 14 days, transduction efficiency was evaluated measuring
the
percentage of cells expressing the V822. Transduced T cells were sorted
according to the
VO expression on the cell surface (Figure 9a).
The functional avidity of HD12-transduced edited T cells was assessed by co-
culture with
.. the EBV- cell line harbouring the HLA-C*07:02 allele pulsed either with the
NYESO-1
peptide as a negative control or with decreasing concentrations (from 40 pg to
0.4 pg) of the
peptide 103 (E:T ratio=1:1). Ability of TCR-transduced T lymphocytes to
recognise target
cells was evaluated by cytofluorimetric analysis determining the expression of
CD107a on
CD8 T cells. Results showed that HD12-transduced edited T cells specifically
recognise
target cells expressing the HLA allele of interest even at a peptide
concentration of 0.4 pg
(Figure 9b).
For testing HD13 and HD14-derived TCRs, which recognise an HLA-A*02:01-
restricted
epitope (peptide 13), activated T lymphocytes isolated from one healthy
individual were
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transduced with the newly produced lentiviral vectors. Transduction efficiency
for HD13
could not be measured due to the absence of an antibody recognising its
specific V13.
Transduction efficiency of HD14-transduced T cells (either with TCR TRAV12-
2*01 WT or
with TCR TRAV12-2*02 WT) was measured by evaluating the percentage of V13
expression
on CD4 and CD8 T cells (Figure 11a).
T cells transduced with HD13 and HD14-derived TCRs were tested for their
functional avidity
in two distinct co-culture experiments. In the first experiment, HD13 TCR
transfer T cells and
HD14 TCR transfer T cells (either the one harbouring the TRAV12-2*01 WT gene
or the
TRAV12-2*02 WT gene) were co-cultured with T2 cells pulsed with WT1 pool or,
as a
.. control, an unrelated pool. In the second experiment, HD13 TCR transfer T
cells and HD14
TCR transfer T cells (harbouring the TRAV12-2*01 WT gene) were co-cultured
with T2 cells
pulsed with subpools 1 and 14 (both containing the peptide 13) and subpool 6
(as a negative
control). As a readout we measured the expression of CD107a and/or IFNy
secretion on
CD8 T cells by cytofluorimetric analysis. We observed a specific recognition
of the target
cells pulsed with the WT1 pool both in T cells transduced with HD13 and HD14-
derived
TCRs (Figure 10a and Figure 11b) and the specific recognition of subpools 1
and 14 (Figure
10b and Figure 11c). Furthermore, HD14-derived TCRs (either the one harbouring
the
TRAV12-2*02 WT gene or the TRAV12-2*02 mut gene) were used in the TCR editing
approach in T cells from one healthy donor. After 14 days, transduction
efficiency was
evaluated measuring the percentage of cells expressing the V1312 (Figure 12a).
To determine the ability of WT1-edited T cells expressing HD14-derived TCRs
(TRAV12-
2*02 WT gene or the TRAV12-2*02 mut gene) to kill primary leukemic blasts, we
evaluated
the expression of active Caspase 3 in target cells upon 6 hour co-culture with
T
lymphocytes. As control, we included co-cultures of unrelated effector cells
with the leukemic
blasts and target cells cultured without effectors. Results showed the ability
of HD14-edited
T cells to recognise primary leukemic blasts at the different effector to
target ratios used
(Figure 12b).
Discussion
The possibility to redirect T cell specificity against antigens expressed by
tumor cells,
through genetic manipulation, has opened up a new therapeutic window for
cancer
immunotherapy. In particular, the recent string of impressive clinical results
obtained with
CAR-redirected T cells (June et al. (2015) Science Translational Medicine 280-
287) have
significantly raised expectations among the scientific community, patient
associations,
pharma and general public. The full exploitation of this strategy largely
relies on the
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identification of receptors specific for relevant tumor antigens. Ideally,
tumor antigens must
be molecules differentially expressed by tumor cells and healthy tissues,
highly
immunogenic, and possibly involved in cancer development and/or progression.
WT1 is a
very attractive target for cancer immunotherapy, and was ranked first in a
list of 75 cancer
antigens within a National Cancer Institute prioritisation project (Cheever
(2009) Clin. Cancer
Res.15: 5323-5337).WT1 is overexpressed by cancer cells 10 to 1000 fold more
than by
healthy tissues (Inoue (1997) Blood 89: 1405-1412), and it is overexpressed in
many
different hematological malignancies, including acute myeloid and
lymphoblastic leukemias
and myelodisplastic syndromes, and by several solid tumors, such as lung
cancer, breast
cancer, esophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma,
pancreatic
cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma,
osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma,
rabdomyosarcoma
(Haruo Sugiyama (2010) Jpn. J. Clin. Oncol. 40: 377-387). Vaccination against
WT1 has
resulted in objective antitumor responses in some cancer patients (Van
Driessche et al.
(2012) Oncologist 17: 250-259). More recently, clinical trials con WT1-
specific T cells,
isolated, expanded and adoptively transferred in patients with acute leukemia
proved safe
and mediated antileukemic activity (Chapuis et al. (2013) Sci Trans! Med 5:
174ra27).
However, the low frequency of high-avidity T cells naturally reactive against
WT1 has limited
up to now the full exploitation of this antigen in adoptive T cell therapy.
The identification of WT1-reactive T cells, and of the genetic sequences of
WT1 specific
TCRs, opens up several novel therapeutic opportunities.
The TCR genetic sequences can be used in their natural forms, or modified, for
example by
murinisation of the constant TCR regions, or by cystein modification of the
human TCR
constant regions, to facilitate proper pairing of the TCR chains, or by codon
optimisation of
the genes, to modify their level of expression.
Natural or modified TCR genes might be transferred in specific T cell subsets,
including CD4
and or CD8, naive, memory stem T cells, central memory, effector memory or
effector cells,
or in other cellular subsets such as to promote a different length of
persistence and different
functions in the engineered cells in vivo. The TCR genes could be also
transferred in T cell
subsets with different polarisation, such as Th0/Tc0, Th1/Tc1, Th2/Tc2, Th17,
Th22 or
others, depending on the cytokine milieu most proper to target each possible
tumor type.
Furthermore these genes, or chimeric genes designed to include the antigen-
specific regions
of the TCR, can be transferred in other cellular subsets, including
gamma/delta T cells, NK
cells, NKT cells, hematopoietic stem cells or other cells, to obtain the
therapeutic effect. It is
furthermore envisaged that natural or modified molecules designed to include
the antigen-
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specific regions of the TCR, could be engineered or coupled to non cellular
substrates such
as nanoparticles, exosomes, or others, or might be used as soluble molecules,
alone or
coupled to other molecules such as toxins or antibodies, thus exploiting their
ability to
recognise tumor cells, thus conferring tumor specificity to cytotoxic
compounds.
Genetic transfer of a novel TCR, such as the TCRs described herein, into T
lymphocytes,
suffers some limitations intrinsic to TCR biology. Specifically, the tumor-
specific alpha and
beta TCR chains are expressed in lymphocytes that already bear an endogenous
TCR on
the cell surface. Gene-modified cells thus express at least two different TCRs
that compete
for binding to the CD3 complex, leading to mutual TCR dilution and reduced T
cell avidity
and anti-tumor efficacy (Heemskerk, M.H. (2007) Blood 109: 235-243).
Furthermore, since
TCRs are heterodimers, the alpha and beta chains of the endogenous TCR might
mispair
with the respective alpha and beta chains of the transgenic TCR to produce new
hybrid
receptors, with unpredictable and potentially harmful specificities (Bendle,
G.M. (2010)
Nature Medicine 16: 565-570; van Loenen, M.M (2010) Proceedings of the
National
Academy of Sciences of the United States of America 107: 10972-10977). These
limitations,
that represent major concerns in TCR gene transfer-based adoptive
immunotherapy, both in
the autologous and in the allogeneic settings, can be addressed by several
strategies,
specifically designed with the aim of increasing TCR expression and fostering
the correct
pairing between tumor specific TCR chains. These strategies include
murinisation of the
constant regions (Cohen C. J. (2006) Cancer Research 66: 8878-8886) the
cystein
modification of the constant regions of the tumor specific TCR genes (Kuball
J. (2007) Blood
109: 2331-2338), or the addition of siRNA designed to limit the expression of
the
endogenous TCR genes (Okamoto S (2009) Cancer Research 69: 9003-9011). Our
group
demonstrated that combining artificial nucleases, such as zinc finger
nucleases (ZFNs),
TALENs or CRISPR/Cas, designed to target the constant regions of the
endogenous TCR
genes (TRAC and TRBC), it is possible to obtain the permanent disruption of
the
endogenous TCR alpha and/or beta chain genes, thus allowing full expression of
the tumor
specific TCR. This process, known as the TCR gene editing proved superior to
TCR gene
transfer in vitro and in vivo (Provasi E., Genovese P. (2012) Nature Medicine
18: 807-15;
Mastaglio S. et al. (2017) Blood 130: 606-618). In addition, the genome
editing technology
allows fostering of the targeted integration of a genetic cassette, inclusive
of the tumor-
specific TCR genes and promoter regions, into the endogenous gene disrupted by
the
artificial nucleases (Lombardo A. (2007) Nature Biotechnology 25: 1298-1306).
Finally, the genome editing technology allows the genetic disruption of
multiple genes in a
single cell: it can thus be envisaged that TCR gene editing could be coupled
with the
nuclease-based disruption of additional genes in the target cell, with the aim
of modifying the
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persistence, expansion, activity, resistance to
exhaustion/senescence/inhibitory signals,
homing capacity, or other functions of the WT1-specific cellular product.
Thus, based on a
single antigenic specificity, we might envisage a wide array of therapeutic
approaches, each
one tailored to the specific tumor type and tumor environment.
EXAMPLE 2
Materials and methods
Isolation of WT1-specific T cells from patient samples
Bone Marrow Aspirate Samples of 3 patients (Pt) diagnosed with Acute Myeloid
Leukemia
who underwent Allogeneic Hematopoietic Transplantation, harvested and
cryopreserved at
the institutional BioBank facility according to the Declaration of Helsinki,
were thawed in X-
VIVO 15 (Euroclone/Lonza) supplemented with Human Serum 5%,
Penicillin/Streptamycin
1% and Glutamine 1%. A few hours after thawing, samples were washed in
Phosphate
Buffered Saline (w/o Ca and Mg) supplemented with EDTA and Fetal Bovine Serum
10%
and subsequently incubated in a total volume of 50 pl with Dasatinib 50 nM for
30 minutes.
After incubation, and without washing, the samples were stained with HLA*0201-
restricted
APC-conjugated dextramers loaded with the VLDFAPPGA (SEQ ID NO: 72) (WT1)
epitope
(ImmuDex) and incubated for 1.5 h on ice.
2 different approaches were tested to isolate WT1-specific T cells:
1. 100 Cells from Patient 1 were directly sorted using a BD FACS Aria cell
sorter in
reverse transcription buffer (SmartScribe; Takara Clontech) in a 1.5 ml
Eppendorf
tube. Afterwards, the sample was heated at 65 C for 2 min followed by 5 min on
ice
and TCRO gene-specific cDNA synthesis was performed (Ruggiero E. et al. (2015)
Nat. Commun. 6: 8081).
2. 500000-2000000 cells from Patients 1, 2, and 3 were stained with anti-APC
magnetic
microbeads (Miltenyi Biotec) according to the manufacturer's instructions and
positively selected using the MACS system (Miltenyi Biotec). The positive
fraction,
enriched in WT1 VLDFAPPGA (SEQ ID NO: 72) specificities, was then cultured in
U-
bottom wells pre-coated with anti-CD3 and anti-CD28 monoclonal antibodies (1:2
ratio) in X-VIVO 15 (Euroclone/Lonza) supplemented with Human Serum 5%,
Penicillin/Streptamycin 1%, Glutamine 1%, IL-2 60 !Wm!, IL-7 5 ng/ml and IL-15
5
ng/ml. Medium was changed every 3-4 days and cells split if confluence was
reached.
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TCR repertoire sequencing
RNA was extracted from WT1-enriched T cells of Patients 1, 2 and 3 by using
the Arcturus
Pico Pure RNA extraction kit (Life Technology). Complementarity determining
region (CDR)
3 sequences of the WT1-specific T cells were amplified by using a modified
RACE approach
(Ruggiero E. et al. (2015) Nat. Commun. 6: 8081). Samples were sequenced by
using an
Illumine MiSeq sequencer and CDR3 clonotypes identified using the MiXCR
software
(Bolotin, DA et al. (2015) Nature Methods 12: 380-381).
Results
The enrichment in WT1 specificities was detected in each individual patient
analysed. For
Patient 1, anti-VLDFAPPGA (SEQ ID NO: 72) (WT1) enrichment occurred at three
different
stages of stimulation (2 weeks and 1 month after first stimulation, 1 month
after second
stimulation), detected by Dextramer staining at flow cytometry (Figure 13a).
For Patient 2
and Patient 3, 2 growing colonies specific for WT1 were detected, one for each
patient, as
assessed by APC-conjugated Dextramer at flow cytometry (Figure 13b).
TCRO sequencing of WT1-specific T cells highlighted the increasing
predominance of
defined CDR3 clonotypes for both TCR chains in each patient analysed (Figure
14a-d).
All publications mentioned in the above specification are herein incorporated
by reference.
Various modifications and variations of the described invention will be
apparent to those
skilled in the art without departing from the scope and spirit of the
invention. Although the
invention has been described in connection with specific preferred
embodiments, it should
be understood that the invention as claimed should not be unduly limited to
such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the
invention which are obvious to those skilled in cell biology, immunology,
immunotherapy,
molecular biology, oncology or related fields are intended to be within the
scope of the
following claims.
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